Fig. 4

DRAM1 suppresses growth factors stimulated rpS6 phosphorylation. a HEK293T cells were transfected with oligos of negative control (NC), DRAM1 siRNA 1 (D1) or DRAM1 siRNA 2 (D2) with the final concentration of 50 nM for 48 h. The mRNA levels were detected with qRT-PCR and normalized to β-actin. Data represent mean ± SEM of combined data from three independent experiments. b HEK293T cells were transfected with negative control or DRAM1 siRNA for 24 h, and then starved for another 18 h before stimulated with IGF-1 (5 ng/ml), EGF (50 ng/ml) or serum (10%) for 10 min. The protein levels of p-rpS6 (S235/236, S240/244), rpS6 and β-actin were detected with immunoblotting. c HEK293T cells were transfected with FLAG empty vector or FLAG-DRAM1 plasmids for 24 h, or the cells were treated with DMSO, MK-2206 (5 μM) or LY2584702 (10 μM) for 24 h, respectively. The protein levels of p-rpS6 (S235/236, S240/244), rpS6, LC3, FLAG and β-actin were detected with immunoblotting. ****p < 0.001 vs the NC group