Phosphorylation of protein targets of PKA and AKT/mTOR signalling pathways in murine ATC7 adrenocortical cells. (A) Phosphorylated mTOR, S6K1, rpS6 and CREB were detected in cells treated for 15′ with increasing concentrations of ACTH. (B) Phosphorylated AKT, mTOR, rpS6 and CREB were detected in cells treated for 2′ to 1 h with PKA hormonal activator ACTH or for 15′ with either the specific PKA activator 8-Br-cAMP or the PI3K/AKT/mTOR pathway hormonal activator insulin. (C) Phosphorylated mTOR, rpS6 and CREB were detected in cells treated in various combinations with ACTH, PKA inhibitor H89 and mTORC1 inhibitor rapamycin. (D) Corresponding phosphorylation signals of mTOR, rpS6 and CREB were quantified (values ± SEM). Values represent relative band density over tubulin expressed as a percentage of the mean of the control. Total mTOR, S6K1, rpS6 and CREB protein signals were unaffected by these short-time treatments (not shown). n = 4; *P < 0.05. Vhl, vehicle; 8Br, 8-Br-cAMP; Ins, insulin; Rapa, rapamycin.