T-I cells were pretreated without or with rapamycin (20 nM) for 1 h prior to treatment for 30 min with insulin (1μg/ml). Control groups were treated with vehicle (DMSO). Cells were lysed and subjected to Western blot analysis using phosphorylation site-specific antibodies for RPS6KB1(Thr389), RPS6KB1(Thr421/Ser424), RPS6 (Ser235/236) and EIF4EBP1(Thr37/46). The levels of RPS6KB1 or RPS6 protein were used as internal controls. Blots are representative of one experiment, and the graphs represent the mean of three experiments. Error bars represent mean ± SE. ***, P< 0.001 vs. control. c represent significant differences compared with insulin (c, P < 0.001). INS= insulin; R= rapamycin