Figure 5

Western blot characterizing the effect of naltrindole, PP1, or AG1478 on tyrosine phosphorylation of the δ-OR by DSLET (1 μM for 5 minutes). HEK-δ-OR cells were serum-starved overnight prior to exposure to DSLET. In some instances, cells were exposed to PP1 (10 μM) or AG1478 (10 μM) for 60 minutes prior to DSLET. After agonist exposure, cell lysates were prepared and the δ-OR was immunoprecipitated as described in the methods section using the anti-FLAG antibody. Tyrosine phosphorylation of the δ-OR was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to the tyrosine-phosphorylated forms of the δ-OR. The immunoblot is representative of an experiment that was repeated three times.