A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture.

Talian, John C.; Olmsted, J.B.; Goldman, Robert D.

doi:10.1083/jcb.97.4.1277

Cited by 184 publications

(96 citation statements)

References 13 publications

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“…To generate rabbit polyclonal anti-VHL antibody, GSThuman VHL (54 ± 213 a.a.) was expressed in Escherichia coli, puri®ed by SDS polyacrylamide gel electrophoresis, and used as an antigen. The antibodies were a nity puri®ed with thioredoxin-fused VHL protein produced in E. coli, following the method described previously (Talian et al, 1983).…”

Section: Antibodiesmentioning

confidence: 99%

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth

Hirai

Kawakami

et al. 2001

Oncogene

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In spite of the general recognition of von Hippel-Lindau (VHL) as a tumor suppressor gene, the physiological and pathological importance of VHL protein in cell growth regulation and tumorigenesis remains unclear. Here we show that in normal human renal proximal tubule epithelial cells (RPTEC), the steady-state amount of VHL protein is strictly regulated by cell density. The cellular VHL content is more than 100-fold higher in dense cultures than in sparse cultures. The increase in VHL protein at high cell density was also observed for NIH3T3 ®broblasts, suggesting the generality of the phenomenon. The growth rates of renal cell carcinoma cells lacking an intact VHL gene and their derivatives with wild-type or mutant VHL expression vector do not di er signi®cantly when they are growing in log-phase. Importantly, however, there is a di erence when they reach con¯uency: cells lacking wild-type VHL grew continuously, while cells expressing exogenous VHL protein showed relatively limited cell growth. Using an ecdysone-inducible VHL expressing cell line, we also show that the growth inhibition at high cell density can be released by attenuating the VHL expression. Taken together, we propose that VHL protein functions as a growth suppressor at high cell density, and this might be the basis of the tumor suppressor function of VHL.

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Section: Antibodiesmentioning

confidence: 99%

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth

Hirai

Kawakami

et al. 2001

Oncogene

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“…All antisera were affinity purified using the respective antigens immobilized by transfer to nitrocellulose paper (66). The bound IgG was eluted either in PBS, pH 7.4, warmed to 56°C (20) or with 0.2 M glycine-HCl (pH 2.8) at 4°C (66).…”

Section: Antibodies and Immunoblottingmentioning

confidence: 99%

“…The bound IgG was eluted either in PBS, pH 7.4, warmed to 56°C (20) or with 0.2 M glycine-HCl (pH 2.8) at 4°C (66).…”

Section: Antibodies and Immunoblottingmentioning

confidence: 99%

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

Drenckhahn

Dermietzel

1988

The Journal of Cell Biology

229

134

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Abstract. In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brushborder cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the ll0-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrooflet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actinactivated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and a-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the ~t-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and ct-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the ll0-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.T HE resorptive surface of the intestinal epithelium, called brush border, is increased 15-30-fold by numerous microvilli that are 1-2 lxm long and 100 nm in diameter (64). Each microvillus is supported by an axial bundle of actin filaments that are in parallel alignment and display identical polarity (51, 53, 59; for review see references 8, 52). The microvillus core bundle is attached to the surrounding membrane by periodically arranged lateral bridges that consist of a complex of a rod-shaped ll0-kD protein and calmodulin (13,31,37,46,47,69). The actin filament core bundle is extensively cross-linked by two proteins, fimbrin (68 kD) and villin (95 kD; 2, 4, 5, 6, 25, 55). In the presence of Ca ++ (>10 -6 M), villin causes fragmentation of the core bundle whereas the bundling activity of fimbrin appears to be independent of Ca++ (9,17,48,49,55). Villin is also present in the rootlets (23). In contrast to the core bundle, the rootlets contain tropomyosin (21). There is a report indicating that the muscular Z-line protein, ~t-actinin, may also be present in the rootlets and absent from the microvilli (27).The spac...

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“…Antibodies of defined specificity were purified from the immune lgG fractions either by the blot-affinity purification procedure of Olmsted (32) or by affinity purification on a column of kinesin immobilized on Sepharose. In the former method, the antibody was eluted from the nitrocellulose paper with 3 M potassium thiocyanate at pH 7.5, followed by dialysis against PBS.…”

Section: Purification Of Antibodiesmentioning

confidence: 99%

Localization of kinesin in cultured cells.

Neighbors

Williams

McIntosh

1988

The Journal of Cell Biology

100

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Abstract. Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of '~120 kD in extracts from bovine brain, PtK~ cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of '~120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of ",,115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK, cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesinpositive structure as a primary cilium. PtK~ cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK~ cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.

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A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture.

Cited by 184 publications

References 13 publications

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study.

Localization of kinesin in cultured cells.

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