Figure 3.

Knockdown of RACK1 increases Bcl-xL protein level. (a) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Bcl-xL protein was normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). **P<0.01 (Student’s t-test). (b) HT1080 cells were transfected with siRNAs against control or RACK1 in combination with or without Beclin1 siRNA (100 pmol). After 48 h, these cells were analyzed by immunoblotting using the indicated antibodies. (c) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h. Extracts of siRNA-transfected cells were pretreated with rapamycin 1 μM for 24 h, followed by immunoblot analysis using the indicated antibodies. (d) Extracts obtained from cells transfected with each siRNA (20 pmol) were immunoprecipitated using Bcl-xL and Beclin1 antibodies. Total cell lysates and the immunoprecipitates were resolved and subjected to immunoblot analysis using the indicated antibodies