Figure 2.
Effect of chronic morphine on the phosphorylation of noncomplexed β-arrestin. (A) Autoradiographic analysis of 32P incorporation into LMMP tissue obtained from opioid naïve (lane 1) and chronic morphine-treated (lane 2) guinea pigs was processed and analyzed as described for Fig. 1 except that anti-β-arrestin antibodies were used for immunoprecipitation. Panel is representative of four experiments. (B) Western blot analysis of LMMP membranes obtained from opioid naïve (lane 1) and chronic morphine-treated guinea pigs (lane 2) using anti-β-arrestin antibodies. Lane 3 represents Western analysis of LMMP membranes obtained from chronic morphine-treated tissue using preadsorbed anti-β-arrestin antibodies. Panel is representative of four experiments. All procedures were performed as described in Materials and Methods. The β-arrestin antibody used was generated against the 75-aa C-terminal domain of β-arrestin2 (≈80% homologous to the corresponding region of β-arrestin1). Furthermore, because this antiserum was polyclonal, it would undoubtedly contain antibody generated against multiple epitopes. It is thus highly unlikely that it differentiates between β-arrestin1 and -2 when used for immunoprecipitation vs. Western analyses.