Fig. 5

In absence of menin an additional treatment with rapamycin reactivates mTORC2 signaling. Menin+/+ and menin−/− MEFs, were serum-starved for 24 h and then stimulated with PDGF-BB (20 ng/ml) for 30 min or as specified, with or without pretreatment with the mTOR inhibitor rapamycin (100 nM) for 24 h + 1 h (a), or in combination with PI3 kinase inhibitor wortmannin (0.2 μm) (b). In menin−/− cells rapamycin retreatment markedly increases mTORC2 signaling (a). Enhanced rapamycin-induced p-Akt occurs downstream of PI3K (b). Menin+/+ MEFs were serum starved and stimulated with PDGF-BB for 30 min after pretreatment with Mek1/2 inhibitor CI-1040, PI3K inhibitor wortmannin (0.2 μm), Ca2+ chelators BAPTA-AM (BA, 10 μM) or EDTA (ED, 2 mM) for 30 min, and rapamycin (100 nM) for 24 h, as indicated (c). Total cell lysate were analyzed by immunoblotting with antibodies specific for menin, Akt phosphorylated on S473 and T450, as well as NDRG1, Rictor, S6 and eIF4B phosphorylation, and the expression of βactin. The relative protein phosphorylations were quantified for a representative experiment