Western blots were performed on a subset of heroin (N=32) and control (N=15) human subjects, and on dorsal striata from the rodent self-administration study, as previously described(32) and probed with primary antibodies directed against the following proteins: MOR (GeneTex, Inc., Irvine, CA); ERK1/2, ELK1 and phospho-ELK1(Ser383)(Cell Signaling Technology, Danvers, MA for human samples and Santa Cruz Biotechnology, Santa Cruz, CA for rat); βarrestin2 (Santa Cruz Biotechnology); and MEK1 (Invitrogen, Carlsbad, CA). Proteins were analyzed using ImageJ software(33, 34) and normalized to total protein levels(35) obtained with the Memcode Reversible Protein Stain Kit (Thermo Scientific). Logarithmic transformations were performed to render normal data distribution. A general linear stepwise regression was used to calculate statistical significance and identify covariates (brain pH, gender, age) using JMP software (SAS Institute, Cary, NC). Two-tailed t-tests were used when no covariates were found. Spearman correlations were calculated to assess the relationship between protein levels and heroin toxicology.
