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Volume 1, Issue 7, Pages 959-968 (June 1998)
The von Hippel-Lindau Tumor Suppressor Protein Is Required for Proper Assembly of an Extracellular Fibronectin Matrix Michael Ohh, Robert L. Yauch, Kim M. Lonergan, Jean M. Whaley, Anat O. Stemmer-Rachamimov, David N. Louis, Brian J. Gavin, Nikolai Kley, William G. Kaelin, Othon Iliopoulos Molecular Cell Volume 1, Issue 7, Pages (June 1998) DOI: /S (00)
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Figure 1 p200 Specifically Coimmunoprecipitates with pVHL
(A and B) 786-O renal carcinoma cells stably transfected with a plasmid encoding HA-epitope-tagged wild-type pVHL ([A], lanes 6–10; [B], lane 2), pVHL Y98H ([B], lane 3), pVHL W117R ([B], lane 4), pVHL R167W ([B], lane 5), or the backbone expression plasmid ([A], lanes 1–5; [B], lane 1) were labeled with 35S-methionine, lysed, and immunoprecipitated with the indicated antibodies (A) or anti-HA antibody (B). Bound proteins were resolved by electrophoresis in a 7.5%–15% discontinuous SDS–polyacrylamide gel and detected by fluorography. Recovery of the pVHL species in (B) was confirmed by anti-HA immunoblot analysis (lower). Asterisk indicates background band (B). Molecular Cell 1998 1, DOI: ( /S (00) )
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Figure 2 p200 Is Fibronectin
(A) 786-O renal carcinoma cells stably transfected with a plasmid encoding HA-epitope-tagged wild-type pVHL (lanes 4–6), pVHL R167W (lanes 7–9), or the backbone expression plasmid (lanes 1–3) were labeled with 35S-methionine, lysed, and immunoprecipitated with the indicated antibodies. Bound proteins were resolved by electrophoresis in a 7.5% SDS–polyacrylamide gel and detected by fluorography. (B) Partial proteolytic peptide mapping of the upper and lower radiolabeled p200 bands in (A) lane 5 (anti-HA) and lane 6 (anti-fibronectin), following digestion with 50 or 500 ng of V8 protease as indicated. (C) 786-O renal carcinoma cells stably transfected with a plasmid encoding HA-epitope-tagged wild-type pVHL (lanes 4–6) or the backbone expression plasmid (lanes 1–3) were lysed and immunoprecipitated with the indicated antibodies. Bound proteins were resolved by electrophoresis in either 7.5% or 15% SDS–polyacrylamide gels and detected by anti-fibronectin or anti-HA immunoblot analysis, respectively. Molecular Cell 1998 1, DOI: ( /S (00) )
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Figure 3 Colocalization of pVHL and Fibronectin in Cells
(A) Immunofluorescence staining of 786-O renal carcinoma cells stably producing HA-wild-type pVHL using mouse anti-HA monoclonal antibody (left) and rabbit anti-fibronectin sera (middle). Bound antibody was detected using FITC-conjugated anti-mouse and rhodamine-conjugated anti-rabbit antibodies, respectively, and a confocal microscope. Overlay of VHL and fibronectin images is shown in the right panel. (B) 786-O renal carcinoma cells ectopically producing HA-wild-type pVHL were disrupted by hypotonic lysis and dounce homogenization. Nuclear (N), cytoplasmic (C), and membrane (M) fractions were prepared and immunoprecipitated with anti-HA (lanes 3–5) and anti-fibronectin (lanes 6–8) antibodies. In parallel, whole cell extracts were prepared and similarly immunoprecipitated with control (lanes 1 and 10), anti-HA (lane 2), and anti-fibronectin (lane 9) antibodies. Specifically bound proteins were detected by anti-fibronectin (upper) or anti-HA (lower) immunoblot analysis. (C) 293 human embryonic kidney cells were fractionated as in (B) and immunoprecipitated with an anti-VHL monoclonal antibody (IG32) (lanes 1–4) or a control antibody (PAb419) (lane 5). Bound proteins were detected by anti-fibronectin immunoblot analysis. Molecular Cell 1998 1, DOI: ( /S (00) )
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Figure 4 pVHL and Fibronectin Form Complexes Prior to Cell Lysis
(A) Unlabeled 786-O renal carcinoma cells ectopically producing HA-pVHL W117R (lane 1) or HA- pVHL (lanes 2–6) were lysed and immunoprecipitated with anti-HA antibody. In parallel, 786-O cells that did not ectopically produce pVHL were radiolabeled with 35S-methionine and lysed. Aliquots of the radiolabeled extract were then added to the unlabeled anti-HA immunoprecipitates in the absence (lanes 1 and 2) or presence of increasing amounts of unlabeled purified fibronectin (lanes 3–6). After an additional 1 hr incubation, the immune complexes were then washed, resolved by SDS–polyacrylamide gel electrophoresis, and detected by fluorography. Both the unlabeled immunoprecipitates and the aliquots of radiolabeled cell extact were derived from a comparable number of cells. (B) 786-O renal carcinoma cells ectopically producing HA-pVHL W117R (lane 1) or HA-pVHL (lanes 2–6) were radiolabeled with 35S-methionine, lysed in the absence (lanes 1 and 2) or presence of increasing amounts of unlabeled purified fibronectin (lanes 3–6; final fibronectin concentration = 28, 56, 83, and 111 nM, respectively), and immunoprecipitated with anti-HA antibody. Bound proteins were detected as in (A). In (A) and (B), lane 6 contained an ∼10-fold molar excess of exogenous unlabeled fibronectin, relative to the amount present in the labeled cell extract, as determined by anti-fibronectin Western blot analysis (data not shown). (NS) indicates nonspecific bands that do not react with anti-fibronectin antibodies in Western blot assays. Molecular Cell 1998 1, DOI: ( /S (00) )
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Figure 5 Renal Carcinoma Cells Display Alterations in Fibronectin Matrix Assembly (A–F) Anti-fibronectin immunofluorescence. A498 (VHL−/−) (A), CAKI-1 (VHL+/+) (B), and 786-O (VHL−/−) (C) renal carcinoma cells, as well as 786-O renal carcinoma cells stably transfected with a plasmid encoding HA-epitope-tagged wild-type pVHL (F), pVHL W117R (E), or the backbone expression plasmid (D), were grown on coverslips for 6 days. Fibronectin deposition was detected by indirect immunofluorescence using a rabbit anti-fibronectin antisera. (G and H) Fibronectin ELISA. A498 (VHL−/−), CAKI-1 (VHL+/+), and 786-O (VHL−/−) renal carcinoma cells, as well as 786-O renal carcinoma cells stably transfected with a plasmid encoding HA-epitope-tagged wild-type pVHL, pVHL W117R, or the backbone expression plasmid (pRc) were grown on a plastic surface for 6 days in 96-well plates. Fibronectin deposition was measured by ELISA as described in the Experimental Procedures. The amount of fibronectin deposited was quantitated from a standard fibronectin curve, and the data is representative of five individual experiments. Error bars indicate one standard deviation. (p < .01 comparing VHL+/+ and VHL−/− cells). Molecular Cell 1998 1, DOI: ( /S (00) )
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Figure 6 Diminished Fibronectin Staining in VHL Nullizygous Mouse Embryos (A) Sagittal sections from VHL+/+ (left) and VHL−/− (right) mouse embryos were stained with either anti-actin (top) or anti-fibronectin (bottom) polyclonal antibodies. Magnification = 40×. (B) Sagittal sections from VHL+/+ (left) and VHL−/− (right) mouse embryos were stained with anti-fibronectin antisera. Shown are higher power magnification (1000×) views of comparable regions. Note the presence of extracellular fibronectin fibrillar arrays in basement membrane regions (arrows). Molecular Cell 1998 1, DOI: ( /S (00) )
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Figure 7 Defective Extracellular Fibronectin Matrix Assembly by VHL−/− Mouse Embryo Fibroblasts VHL+/+ (A and B), VHL+/− (C and D), and VHL−/− (E and F) were grown on coverslips. Fibronectin was detected by indirect immunofluorescence using a rabbit anti-fibronectin antisera (left). Cell number was visualized by DAPI staining. Molecular Cell 1998 1, DOI: ( /S (00) )
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