Hypoxia-Induced ERK Signaling Modulation

UFH-001 cells were exposed to normoxic or hypoxic conditions with or without BCP, at the indicated concentrations, for a total of 16 h. A second set of cells were first exposed to normoxic or hypoxic conditions for 15 ½ h, and then treated for an additional 30 min to JWH-015 at 10 μM (a CB₂ receptor activator). All cells were then placed on ice, washed with ice-cold PBS (10 mM sodium phosphate salts, 120 mM NaCl, pH 7.4), and lysed in RIPA buffer [1% NP-40, 10 mM phosphate buffer, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, 0.5 mM phenylmethyl sulfonyl fluoride (PMSF) and protease inhibitor (Roche Diagnostics), pH, 7.4]. Cell lysates were clarified by centrifugation at 16,000 × g for 15 min at 4°C. Protein concentration of the clarified supernatants was determined using a modification of the Lowry assay [75 (link)]. Equal protein was loaded onto 10% PAGE gels, separated by electrophoresis according to Laemmli et al. [76 (link)], and transferred to nitrocellulose membranes for western blot analysis [77 (link)] using enhanced chemiluminescence (ECL) (GE Healthcare, # RPN2106 or RPN2232). Protein loading was checked by blotting for GAPDH (Cell Signaling, D16H11). Membranes were further probed for total ERK (Calbiochem #442700) or pERK1/2 (Biolabs #9106). Images of the original western blots can be found in the Supplemental material (S1 Raw images).