Inhibition of TORC2 activity by pp242. (A-C) MM cell lines were pre-treated for 30 minutes with increasing concentrations of rapamycin (Rap) or pp242 (pp) and IGF-1 (250 ng/mL) was then added to all cell lines except OPM-2. After 30 minutes additional incubation, protein lysate was immunoblotted for ex-pression of total p70S6kinase (t-P70), phosphorylated P70 (p-P70(T389)), total AKT (t-akt), phosphorylated AKT [p-akt(S473)], total mTOR (t-mtor), phosphorylated mTOR [p-mTOR(S2481)], total NDRG1, phosphorylated NDRG1 (in C), and total ERK or phosphorylated ERK. Arrows in (C) point to the phosphorylated NDRG1 dimer present in control and rapamycin-treated cells. (D) 8226 cells treated with increasing concentrations of pp242 for 1 hour followed by immunoblot for phosphorylated ERK and total ERK. (E) MM1.S and OPM2 cells treated with 100nM of rapamycin for 0, 1, or 24 hours, followed by immunoblot assay for total AKT and S473 phosphorylated AKT.