Figure 5

Eps15 silencing does not affect ErbB2 degradation in GA-treated SK-BR-3 cells. A. SK-BR-3 cells were transfected with siRNA targeting Eps15 or a control siRNA as indicated. Proteins in equal volumes of cell lysate were separated by SDS-PAGE and analyzed by Western blotting, probing with anti-Eps15 and then anti-Hsp70 antibodies. B. Triplicate dishes of SK-BR-3 cells transfected with siRNA targeting Eps15, or a control siRNA, were incubated with 5 μM GA for the indicated times, lysed, and subjected to SDS-PAGE and Western blotting. Equal volumes of each lysate were loaded on the gel. Blots were probed with anti-ErbB2 or anti-Hsp70 antibodies, and then with HRP-conjugated secondary antibodies for detection by chemiluminescence. C. SK-BR-3 cells were transfected and processed as in B, except that blots were probed with AF-680-conjugated secondary antibodies for quantitation of bands using the Odyssey infrared imaging system and the associated software. Results shown are the average of 3 separate experiments, +/- SEM.