Intermediate filament antigens of 60 and 65 kDa in the nuclear matrix of plants: their detection and localization.

Although the presence of a matrix in plant nuclei has been reported, major questions remain about its structural and biochemical features. We have used an intermediate filament antibody of broad specificity to explore whether Daucus carota (carrot) nuclei and nuclear matrices contain intermediate filament/lamin antigens and, if so, where specifically they are localized. SDS-PAGE and Western blotting revealed two bands, at 60 and 65 kDa, that were highly immunoreactive with the intermediate filament antibody (IFA) of Pruss et al. (1981, Cell 27, 419-428). This pattern was observed consistently, not only with carrot cell-free nuclei and nuclear matrices, but also with nuclear preparations from Vicia faba (broad bean) and Pisum sativum (pea). Immunofluorescence studies with whole carrot nuclei localized the IFA antigens to the nucleoplasm and disclosed no accentuated peripheral labeling. Agarose-embedded nuclear matrices showed not only fluorescence throughout the nucleoplasm but also heavy labeling surrounding the nucleoli and suggestions of peripheral labeling. At the ultrastructural level, immunogold results from pre- and postembedment treatments supported the conclusion that IFA antigens occur throughout the nucleoplasm, with possibly a slight concentration at the periphery. These combined results provide substantial evidence that plant nuclei and their matrices possess at least two major intermediate filament antigens with molecular weights characteristic of animal lamins. Whether or not these antigens represent plant lamins, their nonperipheral localization hints at significant differences among the eukaryotic kingdoms in nuclear organization.     

Characterization of 57 kDa statin as a true marker for growth arrest in tissue by its disappearance from regenerating liver

Statin, a 57 kDa nuclear protein, is lost from quiescent fibroblasts in culture when they are induced to enter the cell cycle by feeding with growth factors, or by removal of contact inhibition. In order to investigate changes in statin expression during the transition from a quiescent to a cycling state in situ, we performed 70% partial hepatectomy on rats and analyzed the regenerating liver by immunofluorescence microscopy with antistatin monoclonal antibodies (S44 mAb), and by immunoblotting of liver proteins in cytoplasmic and enriched nuclear/cytoskeletal fractions. Western blot analysis showed that rat hepatocytes in situ contain a nuclear 57 kDa form of statin, as seen in cultured fibroblasts; however additional S44-immunoreactive polypeptides with molecular weights of 53 and 110 kDa are also present in both cytoplasmic and nuclear/cytoskeletal fractions. Immunofluo-rescence microscopy indicates that the proportion of S44-positive hepatocyte nuclei drops to ～60% within 24 hours after hepatectomy, a time period when re-entry of hepatocytes into the cell cycle is first observed. On Western blots of hepatocyte nuclear/cytoskeletal proteins obtained 24 hours after hepatectomy, the 57 kDa form of statin is markedly reduced. These results suggest that, although in liver the S44 antibody recognizes three proteins (53 kDa, 57 kDa, and 110 kDa), the 57 kDa in intact liver, similar to cultured fibroblasts, is the only polypeptide recognized by the statin antibody that disappears when hepatocytes are induced to re-enter the cell cycle from a quiescent state. © 1994 Wiley-Liss, Inc.     

A novel approach to cytotaxonomic and cytogenetic studies in the genus Triturus using monoclonal antibodies to lampbrush chromosomes antigens

Monoclonal antibodies against germinal vesicle antigens from Pleurodeles oocytes crossreact with lampbrush chromosomes of various Triturus species: C36/6, A33/22 and B71/22 bind to most lateral loops, B24/3 labels the spheres, while A1/5 and B81 give a distribution of fluorescent loops which is highly reproducible and species specific. — The antigens involved were characterized by immunoblotting of electrophoretic gels of germinal vesicle proteins and the molecular weights of those that bound to monoclonal antibodies C36/6, A33/22, B24/3 and C3/1 were determined. — The possible relationship between sites immunostained by some monoclonal antibodies and given DNA sequences distributed along the chromosomes is discussed. A new approach to cytotaxonomic and cytogenetic studies through the use of monoclonal antibodies on lampbrush chromosomes is offered, which can give new insight into the molecular mechanisms of speciation and karyological evolution in European newt species.     

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.     

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins.

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.     

Co-localization of cytokinins with proteins related to cell proliferation in developing somatic embryos of Dactylis glomerata L.

The present report provides evidence for co-localization ofcytokinins with cell proliferation-associated nuclear proteins.Somatic embryos of Dactylis glomerata in two stages of developmentare used as a model system comprising both proliferating andinitially differentiated cells. Cytokinins are localized usingantibodies with marked specificity against isopentenyladenine/adenosine(2iP/2iPA) or zeatin/ riboside (Z/ZR). The proliferation-associatednuclear antigen, mitotin, is analysed using a specific monoclonalantibody. The nuclear protein BM28, required for the onset ofDNA replication and for cell division, is identified by an affinity-purifiedpolyclonal antibody. Using double immunofiuorescence labellingwith the antibodies against cytokinins and against each of thenuclear proteins, immunoreaction is observed generally in thesame nuclei of almost all cells in globular embryos and in thenuclei of cells in meristematic areas of the more developedembryos. Only small numbers of individual nuclei positive forboth type of antibodies were found in the surrounding vacuolatedparenchymatous cells. The occurrence of plant antigens homologousto BM28 and mitotin is confirmed by immunoblotting assay. InSDS-PAGE blots the anti-BM28 antibody reacts with a proteinof 58 kDa. The anti-mitotin antibody recognizes several (160,140, 125, 93, and 80 kDa) polypeptides. The data showing nuclearco-localization of cytokinins and proteins with a suggestedrole in the onset of DNA synthesis and in cell division providea new base for further study on the mode of action of cytokininsin cell cycle regulation. Key words: Immunolocalization, cytokinins, nuclear proteins, mitotin, BM28, cell proliferation, somatic embryo(s), Dactylis glomerata     

Analysis of immunocytochemical staining patterns in the antennal system of Drosophila melanogaster

By immunizing mice with homogenized brains, heads, or a mixture of heads and antennae of D. melanogaster, we obtained six monoclonal antibodies (mabs) that bind to the olfactory system of Drosophila with various degrees of specificity. They can be divided into three groups with respect to their staining pattern: (1) The antibodies ca51/2, na21/2, and nb230 label both in the third (olfactory) antennal segment and in the visual ganglia. All of them bind to antennal structures that can be correlated with basiconic sensilla. The antibody ca51/2 labels sensory neurons of these sensilla. In the antenna of the lozenge ³ mutant, which lacks basiconic sensilla, no labeling is present. In Western blots ca51/2 recognizes in the antenna an antigen of 43.5 kDa, which is expressed in the antenna only in the presence of basiconic sensilla. The antibody na21/2 binds to basiconic and coeloconic sensilla, most likely to the apical part of sheath cells. In immunoblots it recognizes in the antenna two antigens of 42.2 kDa and 46.7 kDa. The latter appears to be correlated in the antenna with the presence of basiconic sensilla. (2) The staining pattern of antibody nc10 is associated with the sheath cells of basiconic and coeloconic sensilla. Moreover, nc10 binds to a subset of glomeruli in the antennal lobe. (3) The staining pattern of the antibodies VG2 and I24B5 is restricted to the antenna. I24B5 recognizes coeloconic sensilla and VG2 recognizes both coeloconic and basiconic sensilla. Staining patterns in both cases include sheath cells.     

Analysis of immunocytochemical staining patterns in the antennal system of Drosophila melanogaster

By immunizing mice with homogenized brains, heads, or a mixture of heads and antennae of D. melanogaster, we obtained six monoclonal antibodies (mabs) that bind to the olfactory system of Drosophila with various degrees of specificity. They can be divided into three groups with respect to their staining pattern: (1) The antibodies ca51/2, na21/2, and nb230 label both in the third (olfactory) antennal segment and in the visual ganglia. All of them bind to antennal structures that can be correlated with basiconic sensilla. The antibody ca51/2 labels sensory neurons of these sensilla. In the antenna of the lozenge ³ mutant, which lacks basiconic sensilla, no labeling is present. In Western blots ca51/2 recognizes in the antenna an antigen of 43.5 kDa, which is expressed in the antenna only in the presence of basiconic sensilla. The antibody na21/2 binds to basiconic and coeloconic sensilla, most likely to the apical part of sheath cells. In immunoblots it recognizes in the antenna two antigens of 42.2 kDa and 46.7 kDa. The latter appears to be correlated in the antenna with the presence of basiconic sensilla. (2) The staining pattern of antibody nc10 is associated with the sheath cells of basiconic and coeloconic sensilla. Moreover, nc10 binds to a subset of glomeruli in the antennal lobe. (3) The staining pattern of the antibodies VG2 and I24B5 is restricted to the antenna. I24B5 recognizes coeloconic sensilla and VG2 recognizes both coeloconic and basiconic sensilla. Staining patterns in both cases include sheath cells.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布。结果表明（1）HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40％（55／136）和82％（112／136）。HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;（2）HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81％（133／164）及86％（141／164）。C33c抗原定位于癌细胞及肝细胞的浆内;核心抗原毁定位于癌细胞核中,又可定位于胞浆中。C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细胞在癌组织以弥漫核阳性常见,在癌旁肝组织以胸浆阳性为主;（3）HBxAg在肝细胞肝 癌中的检出率为75％（123／164）,C33c和HBxAg二者同时阳性占63％（103／164）。HCV感染在我国肝细胞肝癌中比较普遍,HCV和HBV重叠感染占相当比例,可能在肝细胞肝癌的发生中起着重要作用。     

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.     

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in hepatocyte nucleus

CD38 is a type II transmembrane glycoprotein found on both hematopoietic and non-hematopoietic cells. It is known for its involvement in the metabolism of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. It is generally believed that CD38 is an integral protein with ectoenzymatic activities found mainly on the plasma membrane. Here we show that enzymatically active CD38 is present intracellularly on the nuclear envelope of rat hepatocytes. CD38 isolated from rat liver nuclei possessed both ADP-ribosyl cyclase and NADase activity. Immunofluorescence studies on rat liver cryosections and isolated nuclei localized CD38 to the nuclear envelope of hepatocytes. Subcellular localization via immunoelectron microscopy showed that CD38 is located on the inner nuclear envelope. The isolated nuclei sequestered calcium in an ATP-dependent manner. cADPR elicited a rapid calcium release from the loaded nuclei, which was independent of inositol trisphosphate and was inhibited by 8-amino-cADPR, a specific antagonist of cADPR, and ryanodine. However, nicotinic acid adenine dinucleotide phosphate failed to elicit any calcium release from the nuclear calcium stores. The nuclear localization of CD38 shown in this study suggests a novel role of CD38 in intracellular calcium signaling for non-hematopoietic cells.     

Localization of antigens PwA33 and La on lampbrush chromosomes and on nucleoplasmic structures in the oocyte of the urodele Pleurodeles waltl: Light and electron microscopic immunocytochemical studies

Monoclonal antibodies A33/22 and La11G7 have been used to study the distribution of the corre-sponding antigens, PwA33 and La, on the lampbrush chromosome loops and nucleoplasmic structures of P. waltl oocytes, using immunofluorescence, confocal laser scanning microscopy and immunogold labeling. The results obtained with these antibodies have been compared with those obtained with the Sm-antigen-specific monoclonal antibody Y12. All these monoclonal antibodies (mAbs) labeled the matrices of the majority of normal loops along their whole length. Nucleoplasmic RNP granules showed a strong staining with the mAbs La11G7 and Y12 throughout their mass, but with the mAb A33/22, they showed only a weak peripheral labeling in the form of patches on their surface. This patchy labeling was confirmed by confocal laser scanning microscopy. Electron microscopy revealed that this patchy labeling might be due to a hitherto undescribed type of submicroscopic granular structure, around 100 nm in either dimension, formed by 10-nm particles. Such granules were observed either attached to the RNP granules or free in the nucleoplasm, but rarely in relation with the normal loop matrices. These 100-nm granules may have a role in the movement of proteins and snRNPs inside the oocyte nuclei for storage, recycling, and/or degradation. Our results also suggest that all the microscopically visible free RNP granules of the nucleoplasm of P. waltl oocytes correspond to B snurposomes. The granules forming the B (globular) loops showed a labeling pattern similar to that of B snurposomes; their possible relationship is discussed.     

Immunoelectron microscope localization of snRNP,hnRNP, and ribosomal proteins in mouse spermatogenesis

We have studied the ultrastructural distribution of heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and ribosomal proteins during mouse spermatogenesis and spermiogenesis by means of specific antibodies and immunocytochemistry. All the above components were detectable from primary spermatocytes until the spermatid elongation phase, when the RNA synthetic activity is known to cease. Ribosomal protein (P1/P2 and L7) labeling disappeared as early as during the acrosome phase, and nucleoli were no longer labeled even during the cap phase. The nucleoplasmic structures labeled with the different anti-nucleoplasmic RNP immunoprobes corresponded, until the acrosome phase, to those previously observed as targets of the same antibodies in the nucleoplasm of somatic cell nuclei. Clusters of interchromatin granules of spermatocyte and early spermatid nuclei exhibit some labeling for hnRNP when compared with nuclei of Sertoli cells or previously analyzed liver or tissue culture cells, where these structural constituents usually remain weakly labeled or unlabeled. In spermatids in step 10, another type of nuclear granule, resembling perichromatin granules, but occurring in aggregates, can be observed. These structural constituents were labeled with antibodies recognizing nucleoplasmic snRNP antigens and therefore suggesting a non-nucleolar origin of these granules. Finally, we have observed nucleoplasmic areas of fibrogranular material, occurring only in primary spermatocytes. These components were labeled with anti-ribosomal protein antibodies but did not contain either hnRNPs or snRNPs. © 1993 Wiley-Liss, Inc.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.     

Lamin A is not synthesized as a larger precursor polypeptide

Isolation of rat liver nuclei in the presence of N-ethylmaleimide (NEM) led to the recovery in the final nuclear matrix of a higher molecular weight form of lamin A. The 2 kDa larger form was identified as lamin A by isoelectric point determination, recognition by an anti-lamin A and C monoclonal antibody and peptide mapping using V8 protease and N-chlorosuccinimide. The 2 kDa extension was tentatively localized to the carboxy-terminus of lamin A. Pulse-chase labeling and immunoprecipitation studies using baby hamster kidney cells showed that lysis of the cells in the presence of NEM allowed the recovery of a stable higher molecular weight form of lamin A. We conclude from these results that NEM prevented the degradation of the native form of lamin A previously thought to represent a higher molecular weight transient precursor form.     

Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.