FIG 1.
Effects of protein phosphatase inhibitors on MEK1/2 phosphorylation in B-RafV600E and K-RasG12C/D tumor cells. (A) Total lysates of SK-MEL 28 cells were incubated with different phosphatase inhibitors for 60 min prior to Western blotting. PPIC3 was diluted 1,000-fold for use as instructed by the manufacturer. PLX4032 (PLX) was used at 20 nM either singly or in combination with PPIC3. Cantharidin, calyculin A, and (−)-p-bromolevamisole oxalate (BO) were used at the indicated concentrations. Dimethyl sulfoxide (DMSO) was the vehicle control. Actin was the control for equal protein loading. pMEK1/2 indicates phosphorylations at Ser217/221 of MEK1 and Ser222/226 of MEK2. (B) Total lysates of SK-MEL 28 cells were incubated with 100 μM cantharidin or 50 nM calyculin A for 60 min, with or without addition of 20 nM PLX4032, prior to Western blotting. (C to F) Total lysates of SK-MEL 28 (C), A375 (D), MiaPaCa-2 (E), and PANC-1 (F) cells were incubated with increasing doses of calyculin A for 15 min or cantharidin for 25 min prior to Western blotting. Right panels, densitometry of phosphorylated MEK1/2 signals normalized for actin.