Inhibition of κ-opioid phosphorylation of ERK1/2. C6 cells were kept in “low MEM” for 48 h before treatment. Opioids and inhibitors were added to the same medium. Cell lysates were prepared, and 15 μg of protein was loaded per lane on 10% sodium dodecyl sulfate-polyacrylamide mini-gels. U69,593 (U69; 10 nM, 10 min) was used to stimulate ERK1/2 phosphorylation. The following inhibitors were applied at the following concentrations and times before U69 treatment: (A) Nor-BNI (1 μM, 1 h), PTX (100 ng/ml, overnight), nifedipine (1 μM, 30 min), U73,122 (1 μM, 30 min), dantrolene (1 μM, 30 min), and BAPTA (50 μM, 30 min) and (B) PMA (1 μM, overnight), GFX (100 nM, 30 min), and PD98059 (1 μM, 30 min). *Significantly greater than all points, p < 0.01 by ANOVA. Data are mean ± SEM (bars) values from three to nine experiments. Data were compiled by densitometric analysis (NIH Image software) and represented as the fold increased intensity as normalized by untreated controls in each experiment. Also shown are representative membranes blotted first with anti-phospho(P)ERK1/2 (top) and then stripped and reblotted with anti-ERK1 (bottom).