Fig. 2. The inability of a membrane-impermeable opioid ligand to block or mimic the effect of NTX on hδOR maturation. HEK-293S-hδOR-FLAG cells were labeled with [35S]methionine/cysteine and then chased for the indicated times in the absence (filled circles) or presence of 50 nM NTX (open circles), 10 µM LE (filled squares) or 50 nM NTX + 10 µM LE (open squares). Cellular membranes were solubilized and the receptors immunoprecipitated and analyzed as described in Figure 1. The graph in (A) describes the time course of appearance and disappearance of the mature Mr 55 000 receptor. Intensity of this species was obtained by densitometric scanning and the values were normalized to the maximum labeling at 4 h of chase in untreated cells. The fluorographs in (B) and (C) correspond to the samples at 2 and 4 h of chase, respectively.