Serum-depleted HepG2 (A, B) and PANC-1 (C, D) cells were pretreated or not in the presence of MNF (1 μM) for 10 min followed by the addition of vehicle, O-1602 (2.5 and 10 μM), or 10% FBS for an additional 10 min. Cell lysates were prepared, separated by reducing SDS-PAGE gel electrophoresis and immunoblotted for total and phosphoactive forms of ERK. A and C, Representative immunoblots; B and D, Phospho-ERK1/2 bands were normalized to total ERK2, and the O-1602-10μM values were set at 1. Data are means of two independent dishes ± range. The migration of molecular-mass markers (values in kilodaltons) is shown on the left of immunoblots. E and F, PANC-1 cells were transfected with control (crtl) or GPR55 siRNA for 48 h, and then were serum-starved for 3 h. ERK1/2 phosphorylation was monitored in cell lysates from vehicle or O-1602 (10 μM)-treated cells. E, Representative immunoblots; F, Signals associated with phospho-active ERK1/2 was normalized to β-actin. Bars represent the mean ± SEM from three independent experiments. *, P < 0.05.