FIGURE 4.

hδOR^Cys-27 impairs maturation and cell surface expression of the hδOR^Phe-27 variant in transiently co-transfected SH-SY5Y neuroblastoma cells. SH-SY5Y cells were co-transfected with HA-hδOR^Phe-27 and Myc-hδOR^Cys-27-FLAG for 24 h using various cDNA ratios as indicated (A and B). As a control, HA-hδOR^Phe-27 was co-transfected with Myc-rLHR-FLAG (δOR/LHR, 1:8) (C and D). After 24 h, cells were analyzed by flow cytometry (B and C) or were lysed and subjected to immunoprecipitation, SDS-PAGE, and Western blotting (WB) using the indicated antibodies (A and D). The values shown in panel B (HA antibody, black bars; cMyc antibody, gray bars) are the means ± S.E. of two-three independent experiments. The GeoMean values were normalized to those obtained from cells that were transfected only with hδOR^Phe-27 or hδOR^Cys-27. The data were analyzed by Bonferroni's Multiple Comparison test after repeated measures one-way analysis of variance before normalization. *, p < 0.05; **, p < 0.01; ***, p < 0.001. In panel C, the shaded curve represents the background signal obtained in the absence of the primary antibody. The black and gray curves correspond to hδOR^Phe-27 in cells co-transfected with rLHR in δOR/LHR DNA ratio of 1:0 and 1:8, respectively. The precursor and mature hδOR forms in panels A and D are indicated with open and closed symbols, respectively, as specified in Fig. 1, and the LHR precursor and mature forms (38) in panel D are indicated with open and closed arrowheads, respectively.