Fig. 5.

Mutation of OPRM1 lysines significantly affects the protein’s stability in NMB cells. a Schematic representations of OPRM1 constructs with 5′ lysines (K). Mutated lysines are shown in bold and italicized. b Relative LUC activity and mRNA levels (expressed as the ratios LUC/β-gal and LUC/LacZ, respectively) in NMB cells transfected transiently with each mutant construct. Bars indicate the standard errors of triplicate LUC assays. c Time course of protein degradation. The relative remaining LUC activity in NMB cells transfected transiently with the constructs shown in a was determined as described in Fig. 3b. d NMB cells cultured in six-well plates were transfected with 2 μg of wild-type (lane 1) or mutated (lanes 2–4) constructs. Forty-eight hours after transfection, immunoprecipitation was performed with anti-luciferase and analyzed by immunoblot using either the antibody to ubiquitin (upper panel) or the antibody to luciferase (lower panel). Numbers on the left (kDa) indicate position of molecular weight marker