Fig. 1. HδOR synthesis and maturation in untreated and NTX-treated HEK-293S-hδOR-FLAG cells. Cells were labeled for 60 min with 150 µCi/ml of [35S]methionine/cysteine and then chased in medium supplemented with 5 mM methionine in the absence (A) or presence (B) of 10 µM NTX for the indicated times. Alternatively, the cells were chased for 4 h in the presence of NTX at the indicated final concentration (C). Cellular membranes were isolated and solubilized in DDM and the receptors immunoprecipitated using the immobilized anti-FLAG M2 antibody as described in Materials and methods. The samples were analyzed by SDS–PAGE and fluorography. Molecular weights of the markers used to calibrate the gels are indicated on the right. The different receptor forms are indicated by arrows and symbols (filled circle, Mr 55 000; open circle, Mr 45 000). The graphs in (A) and (B) describe the time course of appearance and disappearance of the Mr 45 000 and Mr 55 000 receptor species. Intensities of these species were obtained by densitometric scanning and the values were normalized to the maximum labeling of the mature receptor at 4 h of chase in untreated cells. In (C), the intensity of the mature hδOR species (Mr 55 000) was normalized to the maximum labeling of that species and the data were fitted to a sigmoidal dose response equation using the GraphPad Prism program version 2.01.