Fig. 3

SAG interacts with PHLPP1 or DEPTOR and shortens their protein half-lives. (a-e) SAG interacts with PHLPP1 or DEPTOR: DU145 cells were co-transfected with indicated plasmids, cell lysates were prepared and IP with anti-HA Ab, IB with anti-FLAG Ab (a), or IP with anti-FLAG Ab, IB with anti-HA Ab (b). Sub-confluent DU145 cells were lysed and subjected to IP with anti-SAG (c), anti-PHLPP1 (d) or anti-DEPTOR Ab (e), along with IgG control, followed by IB with indicated Abs. (f & g) SAG overexpression shortens protein half-lives of PHLPP1 and DEPTOR: DU145 cells were transfected with HA-PHLPP11or FLAG-DEPTOR, along with the vector control or FLAG-SAG for 48 hrs. Cells were treated with cycloheximide (CHX; 100 μg/ml) to block new protein synthesis for the indicated time periods, followed by IB analysis. (h & i) SAG knockdown extends the protein half-lives of PHLPP1 and DEPTOR: DU145 cells were infected with Lt-SAG, along with Lt-Con for 72 hrs. Cells were then treated with CHX (100 μg/ml) for indicated time periods and subjected to IB analysis. Densitometry quantification was performed using ImageJ software with β-actin as the loading control and plotted (bottom panels)