Effect of cocaine on MOR protein and mRNA expression in PC12 cells. (A) Representative immunoblots obtained from lysates of control (untreated) and continuous (SCT) or intermittent (RIT) 10, 100, 500 μM cocaine treated PC12 cells separated by SDS-PAGE and transferred to nitrocellulose membranes. The top panel represents total MOR expression and the bottom panel shows α-tubulin levels from the same blot. (B) Densitometric analysis of MOR protein levels relative to α-tubulin levels in cocaine-treated cells revealed a significant increase in MOR expression following 500 μM SCT or 100 μM RIT compared to control. (C) Cell lysates were obtained from cells exposed to 500 μM SCT or 100 μM RIT with cocaine for 72 h, followed by 10 μg/mL cycloheximide for 4, 8, 12, 24 and 48 h. Densitometric analysis of MOR protein expression normalized to α-tubulin revealed an approximate half-life of 36.3 h in control (untreated) cells, and a significant change in the slope of protein decay following both cocaine treatment regimens. Protein half-life could not be estimated for RIT, but an increase in the predicted half-life to 85.3 h was calculated for SCT. (D) Quantitative PCR (qPCR) analysis of MOR mRNA levels relative to β-2 microglobulin in control and cocaine treated cells. Relative to control, 100 μM of RIT with cocaine significantly increased MOR mRNA levels while 500 μM of SCT had no effect. Results are representative of at least 5 independent experiments and the data are presented as mean ± SEM (*p<0.05, **p<0.01, ***p< 0.001, **^p=0.001).