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Fig. 4. PDGF stimulates two pools of SFK activities with distinct sensitivity to membrane cholesterol. (A) Purification of CEF. 1% Triton X-100 cell lysates of fibroblasts treated as indicated were subjected to a Dounce homogenisation followed by a sucrose gradient fractionation. Fractions were directly subjected to western blotting with an anti-phosphotyrosine (4G10) and caveolin-specific antibody as indicated. The presence of respective protein as well as the fraction number is shown. (B) PDGF-induced SFK activities in caveolae-enriched (CEF) and non-caveolae (NCF) fractions. CEF (2-4) and NCF (7-9) were pooled and treated as in the Materials and Methods. SFK activities and their association with caveolin were measured by western blotting of immunoprecipitated kinases with pY416Src and caveolin antibody respectively. (C) CO regulates SFK-PDGFR association in CEF. In vitro kinase assay was performed with the immunoprecipitated SFK from CEF. The presence of PDGFR was revealed by re-immunoprecipitation of the labelled proteins using specific antibody (
PR4) as indicated (2nd ip). Antibodies used for immunoprecipitation (ip), cell treatments, SFK, pY416 SFK, heavy chains immunoglobulin (Hc), [32P]SFK and [32 P]PDGFR are indicated. (D) Cholesterol content in CEF. CEF were isolated as described in Materials and Methods from NIH 3T3 cells treated or grown as indicated and from HEK 293 cells expressing Cav3DGV as indicated. Is shown the cholesterol content in CEF relative to the non treated cells (% control). (E) Levels of SFK in CEF and NCF fractions. SFK were immunoprecipitated from two-thirds of CEF and one-quarter of NCF and subjected to western blotting with cst1 antibody. Caveolin-1 and ß 1 integrin levels were detected as specific markers of CEF and NFC respectively and assessed by western blotting of total protein fractions. A quantification of SFK levels is indicated.
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