FIGURE 3.

hδOR^Cys-27 redirects hδOR^Phe-27 precursors to ERAD in stably co-transfected HEK293_i cells. HEK293_i cells constitutively expressing HA-hδOR^Phe-27 were induced with tetracycline to express Myc-hδOR^Cys-27-FLAG for 16 h or were left untreated. A, cells were pulse-labeled with [³⁵S]methionine/cysteine for 40 min and chased for 2 h. Lactacystin (10 μm) or vehicle was added 60 min before labeling. The hδOR^Phe-27 and hδOR^Cys-27 variants were purified from lysates by two-step immunoprecipitation with the HA and FLAG M2 antibodies, respectively, and analyzed by SDS-PAGE and fluorography. B, alternatively, the variants were purified as described above by one-step immunoprecipitation from lysates of non-labeled cells that were treated or not with lactacystin for 6 h at the end of the 24-h induction. The purified samples were subjected to SDS-PAGE and Western blot (WB) analysis with the ubiquitin antibody. The precursor and mature receptor forms are indicated with open and closed symbols, respectively, as specified in Fig. 1. The asterisks depict polyubiquitinated receptors.