Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity

The biomedical research community relies directly or indirectly on immunocytochemical data. Unfortunately, validation of labeling specificity is difficult. A common specificity test is the preadsorption test. This test was intended for testing crude antisera but is now frequently used to validate monoclonal and affinity purified polyclonal antibodies. Here, the authors assess the power of this test. Nine affinity purified antibodies to different epitopes on 3 proteins (EAAT3, slc1a1; EAAT2, slc1a2; BGT1, slc6a12) were tested on samples (tissue sections and Western blots with or without fixation). The selected antibodies displayed some degree of cross-reactivity as defined by labeling of samples from knockout mice. The authors show that antigen preadsorption blocked all labeling of both wild-type and knockout samples, implying that preadsorption also blocked binding to cross-reactive epitopes. They show how this can give an illusion of specificity and illustrate sensitivity-specificity relationships, the importance of good negative controls, that fixation can create new epitopes, and that cross-reacting epitopes present in sections may not be present on Western blots and vice versa. In conclusion, they argue against uncritical use of the preadsorption test and, in doing so, address a number of other issues related to immunocytochemistry specificity testing.     

Effect of antiserum to a 99 kDa polypeptide on the uptake of taurocholic acid by rat ileal brush border membrane vesicles

A 99 kDa polypeptide in rat ileal brush border membrane (BBM), regarded as a component of the active bile acid transport system on account of photoaffinity labeling, has been purified by affinity chromatography and preparative gel electrophoresis and utilized as an immunogen for raising polyclonal antibody. Immune serum, but not preimmune serum, specifically recognized a single band of 99 kDa protein on immunoblots of ileal and renal BBM. In contrast, no reactivity was observed with proteins in jejunal BBM. This polyclonal antibody, compared with preimmune serum and anticytosolic bile acid binding protein (14 kDa) serum, significantly inhibited the Na+ dependent uptake of [3H] taurocholate by BBM vesicles (p less than 0.01). [14C] D-glucose uptake by BBM vesicles was not influenced by the immune serum (p less than 0.01). Thus, these studies provide further support for the specific role of a 99 kDa protein in ileal BBM bile acid transport.     

MAP 4: occurrence in mouse tissues

A polyclonal antiserum to a microtubule-associated protein (MAP) from mouse neuroblastoma cells (MAP 4) was used to examine the distribution of this protein in mouse tissues. Immunoblots of neuroblastoma cell microtubule protein preparations demonstrated that the antiserum reacted with a triplet of proteins at 215,000-240,000 mol wt. Antibodies affinity purified from any of the bands showed cross- reaction with the other bands, indicating these polypeptides were all immunologically related. Antibodies specific to MAP 4 decorated microtubules in cultured murine cells fixed with glutaraldehyde, and diffuse staining was seen following treatment of cells with nocodazole. The antiserum reacted with MAP 4 in extracts of brain, heart, liver, and lung from adult mouse; the triplet in brain was more closely spaced than in the other tissues or neuroblastoma cells. In kidney, spleen, and stomach, only a single band (band 4) was labeled; this band was immunologically related to the triplet and was also present in all tissues positive for the triplet. Skeletal muscle, sperm, and peripheral blood contained no reactive polypeptides. After taxol- induced polymerization, the MAP 4 triplet was preferentially associated with the microtubule pellet whereas band 4 remained in the supernatant. These data indicate that there is tissue specificity in the distribution of MAP 4, and that some tissues contain a polypeptide related to MAP 4 (band 4) that does not bind to microtubules in vitro.     

Schistosoma japonicum: immunoinhibitory studies on hemoglobin digestion using heterologous antiserum to bovine cathepsin D.

Antibody against purified bovine cathepsin D was raised in rabbits, and the polyclonal antiserum was tested to determine its ability to inhibit the hemoglobinolytic activity of the crude enzyme preparation (CEP) from adult Schistosoma japonicum and its effect upon in vitro cultured Schistosoma mansoni schistosomules. The 100,000 g supernatant fraction (CEP) from lyophilized adult worms was preincubated with antiserum and subsequently incubated with hemoglobin. Hemoglobinolytic activity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedures. Five hours of incubation failed to diminish hemoglobin concentration in experimentals, whereas controls treated with preimmune serum displayed hemoglobin degradation. Pepstatin inhibited hemoglobin degradation. Western blot analysis of the CEP revealed a broad band of activity at approximately 45 kDa. Schistosomules incubated in vitro either in the antiserum or pepstatin and subsequently exposed to host erythrocytes showed a marked inhibition of digestive activities. Although structural changes were not evident in the gastrodermis, some perturbation of the tegument was observed. Schistosomules fed host erythrocytes and postincubated in the antiserum displayed increased tegumental perturbation and extensive alteration of the gastrodermis, including dilation of cisternae and membrane disruption. Schistosomules exposed to preimmune serum were normal in all respects.     

Immunochemical Characterization of the β2 Subunit of the GABAA Receptor

To date three β subunits of the GABA_A receptor have been identified in rat brain as a result of cDNA library screening. The β2 subunit has been reported to have a wide distribution in rat brain based on in situ hybridization studies quantifying β2 mRNA. To study the β2 subunit more directly, we have raised a polyclonal antibody to a synthetic peptide representing residues 315–334 of the intracellular loop of the β2 subunit. The antibody, which had been affinity-purified, recognized the β2 peptide but did not immunolabel homologous β1 and β3 subunit peptides, indicating that this antibody is specific for the β2 subunit of the receptor. In western blots of the purified receptor, the antibody recognized a major diffuse band of 54–58 kDa arid exhibited minor labeling of lower-molecular-mass polypeptides. In western blots of cortex homogenate, the antibody exhibited nervous system-specific labeling of a 55-kDa band that comigrated with the 55-kDa band of the purified receptor. Quantitative immunolabeling of this 55-kDa polypeptide permitted direct determination of the relative amounts of the β2 subunit in different brain regions. The brainstem contained the highest relative specific activity of the β2 subunit, followed by the inferior colliculus, olfactory lobe, and cerebellum. Lower levels of immunolabeling were seen in hypothalamus, hippocampus, thalamus, and cortex.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Subcellular distribution of 14-3-3 proteins in the unicellular green alga Chlamydomonas reinhardtii.

A polyclonal antibody was raised against a recombinant Chlamydomonas 14-3-3-beta-galactosidase (beta-Gal) fusion protein and characterized for its epitope specificity towards the corresponding Chlamydomonas 14-3-3 protein by scan-peptide analysis. This antibody recognized four Chlamydomonas polypeptides with apparent molecular masses 32, 30, 27, and 24 kDa, which also reacted with the antiserum depleted of anti-(Escherichia coli beta-Gal) IgG, but not with the corresponding preimmune serum or the antiserum preincubated with purified 14-3-3 proteins. Western-blot analyses performed with the antibody depleted of anti-(beta-Gal) IgG revealed that more or less pronounced levels of 14-3-3 proteins were present in all subcellular fractions of Chlamydomonas reinhardtii except the nuclei. The highest levels of 14-3-3 protein were observed in the cytosol and microsomal fraction. The 30-kDa isoform was predominant in the cytosol, whereas the 27-kDa isoform was prevalent in the microsomes. When microsomal membranes were separated by sucrose-density-gradient centrifugation, Western-blot analysis revealed distinct patterns of 14-3-3 isoforms in the endoplasmic reticulum, dictyosome, and plasma membrane fractions identified by marker enzyme activities. These findings indicate that the four 14-3-3 proteins of C. reinhardtii differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane.     

Immunological characterization of a newly developed antibody for localization of a beta-keratin in turtle epidermis

Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.     

The sequential cleavage of membrane anchored pro-EGF requires a membrane serine protease other than kallikrein in rat kidney

Epidermal growth factor (EGF) is present in kidney membranes as an integral type I precursor protein, enzymatically processed to release immunoreactive materials in urine or incubation medium. The aim of this work was the elucidation of both the anchor of the serine protease activity that processes pro-EGF, and the determination of the steps of the enzymatic processing. Quantification of EGF containing molecules by RIA following gel filtration analysis demonstrated that the membrane precursor is first shed from the kidney membrane principally into a 170-kDa soluble precursor. This entire ectodomain is further processed into a 70-kDa precursor and finally into the mature 5.9 kDa urinary EGF. These species correspond to the ones found in urines. Both shedding and maturation events are clearly realized by membrane anchored serine protease activity, which remains active in detergent. By use of wild-type and knockout mice urines, we found that tissue kallikrein (TK) was not involved in the regulation of this processing.     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Expression of a novel sodium-hydrogen exchanger in the gastrointestinal tract and kidney

Aquaporin CHIP, a 28 kDa channel forming protein, has been proposed to function as water channel in both erythrocyte and kidney proximal tubule. Recently, we have reported that in frog urinary bladder, a model of the kidney collecting tubule, polyclonal antibodies against human erythrocyte CHIP recognize and immunoprecipitate a 30 kDa protein from the epithelial cell homogenate. In the present work confocal fluorescence microscopy was used to determine the cellular and subcellular localization of CHIP28-like proteins in the urinary epithelium. A clear labeling of the apical border was found after Triton X-100 permeabilization. The labeling was distributed throughout the apical domain and not restricted to specific domains of the membrane. The staining was also present in the deeper confocal sections where the fluorescence seems to be localized at the cellular contour. No difference in the labeling patterns was observed between resting and ADH-treated bladder. Specificity of the staining was confirmed by the absence of the labeling pattern when antiserum was preadsorbed on CHIP28 protein immobilized on Immobilon P stripes. Our results suggest that CHIP-like proteins are not proteins inserted in the apical membrane during the antidiuretic response. Moreover, we do not know whether the labeling was due to the presence of CHIP28 itself or an as-yet-unidentified protein sharing immunological analogies with aquaporin CHIP.     

Immunological properties of O2.- generating oxidase from bovine neutrophils

Two antisera have been prepared against the O2.- generating oxidase purified from bovine polymorphonuclear neutrophils (PMNs). The first antiserum was directed against the enzymatically active fraction obtained after isoelectric focusing (pI oxidase), which consisted of a major protein of Mr 65,000 [(1985) Biochemistry 24, 7231-7239]. The second antiserum was directed against the 65 kDa band excised from an SDS-polyacrylamide gel after electrophoresis of the pI oxidase preparation. The pI oxidase antiserum inhibited O2.- generation by PMN cells, PMN membranes and detergent-solubilized membranes. The 65 kDa band antiserum was virtually non-inhibitory against PMN cells; in contrast, it was nearly as potent as the pI oxidase antiserum on PMN membranes and detergent-solubilized membranes. Inhibition of O2.- generation by the pI oxidase antiserum was correlated with the immunoreactivity of four membrane-bound proteins of 65, 54, 18 and 16 kDa; the 65 kDa band antiserum reacted only with the two proteins of 65 and 54 kDa. It is concluded that the 18 and 16 kDa proteins, present in trace amounts in the pI oxidase preparation, are probably potent catalysts of the respiratory burst.     

An antiserum to the C-terminus of the Alzheimer amyloid precursor recognizes a soluble 70 kDa protein

A rabbit antiserum to the C-terminus of the putative brain amyloid precursor was used to probe Western blots of tissue proteins separated by SDS-PAGE. The antiserum specifically labelled a protein of approx. 70 kDa in the Tris buffer-soluble fraction of brain samples from rat, Alzheimer subjects, cases of young and old Down's syndrome, and age-matched controls. The 70 kDa protein was present in low concentrations in human liver and kidney, and was undetectable in human skeletal muscle. The 70 kDa protein may be a metabolite of the amyloid precursor.     

Identification of retinal insulin receptors using site-specific antibodies to a carboxyl-terminal peptide of the human insulin receptor alpha-subunit. Up-regulation of neuronal insulin receptors in diabetes

Insulin receptor-specific polyclonal antipeptide serum was generated against a synthetic pentadecapeptide (residues 657-670) of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina. The affinity-purified antibodies recognized peptide antigen but not keyhole limpet hemocyanin as determined by dot blot analysis and solid phase radioimmunoassay. Addition of either synthetic peptide or the affinity-purified serum had no effect on 125I-insulin binding to placental membranes or to cells in culture. alpha-Subunits of approximately 125 kDa from human placental membranes and liver membranes were labeled by immunoblot analysis with this antiserum. In membranes isolated from human retina and brain, two classes of alpha-subunits of approximately 125 and 115 kDa were detectable. The 115-kDa subunit was neuraminidase resistant whereas the 125-kDa subunit was digested to a band of 115 kDa, indicating that these bands represent peripheral and neuronal receptors, respectively. Analysis of human retinas obtained from type I diabetic donors revealed an increased level of neuronal receptor as compared with normal retinas. These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes.     

Introductory study of an oocyte membrane protein that specifically binds vitellogenin in the crayfish Orconectes limosus

Summary

After solubilization of membranes from vitellogenic oocytes of the crayfish Orconectes limosus, a component with an apparent molecular weight between 28 and 30 kDa that specifically binds vitellogenin, could be identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by electroblotting. Because this component was pronase sensitive, we assume that we are dealing with a protein. A polyclonal rabbit antiserum was produced against this component and its tissue specificity was verified.     

Immunolocalization of AQP9 in liver, epididymis, testis, spleen, and brain

The aims of this study were to determine the cellular and subcellular localization of aquaporin-9 (AQP9) in different rat organs by immunoblotting, immunohistochemistry and immunoelectron microscopy. To analyze this, we used rabbit antibodies to rat AQP9 raised against three different AQP9 peptides (amino acids 267-287, 274-295, and 278-295). In Cos7 cells transfected with rat AQP9, the affinity-purified antibodies exhibited marked labeling, whereas nontransfected cells and cells transfected with aquaporin-8 (AQP8) exhibited no labeling, indicating the specificity of the AQP9 antibodies. Immunoblotting revealed a predominant band of 28 kDa in membranes of total rat liver, epididymis, testes, spleen, and brain. Preabsorption with the immunizing peptides eliminated the labeling. Immunohistochemistry showed strong anti-AQP9 labeling in liver hepatocytes. The labeling was strongest at the sinusoidal surface, and there was little intracellular labeling. Immunoelectron microscopy revealed that the labeling was associated with the plasma membrane of the hepatocytes. In testes Leydig cells exhibited anti-AQP9 labeling, and in epididymis, the stereocilia of the ciliated cells (principal cells) exhibited significant labeling, whereas there was no labeling of the nonciliated cells (basal cells). This was confirmed by immunoelectron microscopy. In spleen strong labeling of cells was observed of leukocytes in the red pulp, whereas there was no labeling of cells in the white pulp. In rat brain, AQP9 immunolabeling was confined to ependymal cells lining the ventricles and to the tanycytes of the mediobasal hypothalamus. Antibody preabsorbed with the immunizing peptide revealed no labeling. In conclusion, AQP9 proteins is strongly expressed in rat liver, testes, epididymis, spleen, and brain.     

Cloning and immunolocalization of a rat pancreatic Na(+) bicarbonate cotransporter.

In the rat, pancreatic HCO(-)(3) secretion is believed to be mediated by duct cells with an apical Cl(-)/HCO(-)(3) exchanger acting in parallel with a cAMP-activated Cl(-) channel and protons being extruded through a basolateral Na(+)/H(+) exchanger. However, this may not be the only mechanism for HCO(-)(3) secretion by the rat pancreas. Recently, several members of electrogenic Na(+)/HCO(-)(3) cotransporters (NBC) have been cloned. Here we report the cloning of a NBC from rat pancreas (rpNBC). This rpNBC is 99% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas, prostate, and a minor clone in kidney]. The longer NBC isoforms are identical to the rat and human kidney-specific forms (kNBC; 1035 aa; 116 kDa) at the approximately 980 C-terminal aa's and are unique (with different lengths) at the initial N-terminus. Using polyclonal antibodies to the common N- and C-termini of rat kidney NBC, a approximately 130-kDa protein band was labeled by immunoblotting of rat pancreas homogenate and was enriched in the plasma membrane fraction. Immunofluorescence and immunoperoxidase light microscopy of rat pancreatic tissue with both antibodies revealed basolateral labeling of acinar cells. Labeling of both apical and basolateral membranes was found in centroacinar cells, intra- and extralobular duct, and main duct cells. The specificity of the antibody labeling was confirmed by antibody preabsorption experiments with the fusion protein used for immunization. The data suggest that rpNBC likely plays a more important role in the transport of HCO(-)(3) by rat pancreatic acinar and duct cells than previously believed.     

Distribution of GLUT3 glucose transporter protein in human tissues.

To investigate the tissue distribution of the GLUT3 glucose transporter isoform in human tissue we produced affinity purified antibodies to the COOH terminus of the human GLUT3. Both antibodies recognize a specific GLUT3 band in oocytes injected with GLUT3 mRNA but not in those injected with H2O or GLUT1, 2, 4, 5 mRNA. This immunoreactive band in GLUT3 injected oocytes is photolabelled by cytochalasin-B in the presence of L- but not D-glucose indicating that it is a glucose transporter. A high cross reactivity between the human GLUT3 antibodies and a 43 kDa cytoskeletal actin band was identified in all oocyte lysates and many human tissues. However, the specific GLUT3 band could be distinguished from the actin band by carbonate treatment which preferentially solubilized the actin band. Using these antibodies we show that GLUT3 is present as a 45-48 kDa protein in human brain with lower levels detectable in heart, placenta, liver and a barely detectable level in kidney. No GLUT3 was detected in membranes from any of 3 skeletal muscle groups investigated. We conclude that a major role of GLUT3 in humans is as the brain neuronal glucose transporter.     

Mammalian-heart adenylate deaminase: Cross-species immunoanalysis of tissue distribution with a cardiac-directed antibody

A sheep antiserum against purified rabbit-heart adenylate deaminase (EC 3.5.4.6) (AMPD) was developed and validated as an immunologic probe to assess the cross-species tissue distribution of the mammalian cardiac AMPD isoform. The antiserum and the antibodies purified therefrom recognized both native and denatured rabbit-heart AMPD in immunoprecipitation and immunoblot experiments, respectively, and antibody binding did not affect native enzyme activity. The immunoprecipitation experiments further demonstrated a high antiserum titer. Immunoblot analysis of either crude rabbit-heart extracts or purified rabbit-heart AMPD revealed a major immunoreactive band with the molecular mass (81 kDa) of the soluble rabbit-heart AMPD subunit. AMPD in heart extracts from mammalian species other than rabbit (including human) was equally immunoreactive with this antiserum by quantitative immunoblot criteria. Although generally held to be in the same isoform class as heart AMPD, erythrocyte AMPD was not immunoreactive either within or across species. Nor was AMPD from most other tissues [e.g., white (gastrocnemius) muscle, lung, kidney] immunoreactive with the cardiac-directed antibody. Limited immunoreactivity was evidenced by mammalian liver, red (soleus) muscle, and brain extracts across species, indicating the presence of a minor cardiac(-like) AMPD isoform in these tissues. The results of this study characterize the tissue distribution of the cardiac AMPD isoform using a molecular approach with the first polyclonal antibodies prepared against homogeneous cardiac AMPD. This immunologic probe should prove useful at the tissue level for AMPD immunohistochemistry.     

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.