Fig. 3

B56γ3 upregulates AKT phosphorylation through targeting PP2A to downregulate p70S6K phosphorylation at Thr389 and attenuate p70S6K-mediated negative feedback loop regulation on growth factor signaling. A Lysates of HEK293 cells co-transfected with expression vector encoding 4HA-B56γ3 and Flag-p70S6K or vector only were immunoprecipitated by anti-FLAG-Sepharose, and the immunocomplexes were analyzed by SDS-PAGE and Western blotting with specific antibodies as indicated. B Lysates of HEK293 cells transfected with expression vector encoding Flag-p70S6K were immunoprecipitated by anti-FLAG-Sepharose, and the immunocomplexes were analyzed for the presence of endogenous B56γ3. C Lysates of HCT116 cells transfected with expression vector encoding 4HA-B56γ3 were immunoprecipitated by anti-HA-Sepharose, and the immunocomplexes were analyzed for the presence of endogenous p70S6K. D In vitro pulldown analysis of recombinant GST or GST-p70S6K interacting with recombinant His-B56γ3 using glutathione Sepharose was carried out and GST-pulldowns were then analyzed by SDS-PAGE and Western blotting with antibodies as indicated. Ten percent of mixed recombinant proteins after pulldown analysis were analyzed in parallel, serving as a loading control. E Lysates of HeLa cells transfected with expression vector encoding Flag-p70S6K and 4HAB56γ3, 4HAB56γ1, 4HAB56α, or empty vector were analyzed by SDS-PAGE and Western blotting with antibodies as indicated. F Lysates of HeLa cells transfected with expression vectors encoding Flag-p70S6K and 4HAB56γ3 treated with or without 500 nM okadaic acid (OA) were analyzed by SDS-PAGE and Western blotting with antibodies as indicated. G Lysates of HeLa cells stably expressing shLuc or shB56γ3 were analyzed by SDS-PAGE and Western blotting with antibodies as indicated. H Lysates of SW480 cells stably expressing shLuc or shB56γ3 stimulated by IGF-1 (50 ng/ml) at the indicated time points were analyzed as described earlier. I Lysates of HCT116 cells stably expressing shLuc or shB56γ3 stimulated by EGF (20 ng/ml) at the indicated time points were analyzed as described earlier. J Lysates of SW480 cells with control shLuc or shB56γ3 pretreated with DMSO or 5 μM LY-2584702 (Adooq Bioscience) in serum-free medium, prior to stimulation with IGF-1 (50 ng/ml) for 60 min in the presence or absence of 5 μM LY-2584702, were analyzed as described previously. K Lysates of HeLa cells with control shLuc or shB56γ3 pretreated with DMSO or 5 μM LY-2584702 in 0.5% FBS medium prior to stimulation with EGF (50 ng/ml) at the indicated time points in the presence or absence of 5 μM LY-2584702 were analyzed as described previously. L Lysates of HCT116 cells with control shLuc or shB56γ3 pretreated with DMSO or 5 μM LY-2584702 in serum-free medium prior to stimulation with EGF (20 ng/ml) for 30 min in the presence or absence of 5 μM LY-2584702 were analyzed as described previously. Representative blots of at least two independent experiments with similar results were shown. The mean relative expression levels of phospho-Akt (Thr308 and Ser473) and phospho-p70S6K (Thr389) of three independent experiments with similar results were quantified as described earlier. The differences were assessed for statistical significance by student’s t test with p value (*(< 0.05), **(< 0.01), ***(< 0.001)). (n = 3)