FIGURE 8.

Effects of the modulation of caveolin-1 and cellular ganglioside levels on c-Src activity. c-Src activity has been measured as autophosphorylation using an immunocomplex kinase assay as described under “Experimental Procedures.” Briefly, c-Src has been immunoprecipitated with a specific monoclonal antibody from cell lysates obtained under the different experimental conditions. Immunoprecipitates have been incubated with 10 μm [γ-³²P]ATP. After incubation, proteins associated with each sample have been separated by SDS-PAGE. The total amount of c-Src associated with each sample has been determined by Western blotting and densitometry (left panels). The amount of radioactivity associated with the band corresponding to c-Src protein has been determined by autoradiography and has been normalized for the total c-Src content associated with each sample (right panels). Data are the means ± S.D. of four independent experiments. Panel A, effect of caveolin-1 silencing in A2780/HPR cells. A2780/HPR cells were treated with siRNA targeting to CAV1 mRNA (gray bars) or with scrambled siRNA as control (white bars). Analysis has been performed 48 h after siRNA administration. *, p < 0.03 versus control. Panel B, effect of ganglioside depletion by treatment with d-PDMP in A2780/HPR cells. A2780/HPR cells were treated with 20 μm d-PDMP (gray bars) or with vehicle (white bars) for 48 h. *, p < 0.02 versus control. Panel C, effect of exogenous gangliosides administration in A2780 cells. Cells were treated with 50 μm GM2 for 48 h or with 50 μm GM3 for 24 and 48 h in serum-free medium as described under “Experimental Procedures.” *, p < 0.01 and °, p < 0.03 versus time-matched controls.