FIGURE 8.
Effect of siRNA-induced depletion of the co-chaperones P23 and CHIP on A2A receptor levels. A and B, HEK293 cells stably expressing the FS2-N-A2A receptor were transfected either with negative control siRNA or with siRNAs targeting P23, CHIP, and HSP90α (as an internal reference, compare Fig. 6). After 48 h, cells were lysed. Aliquots of the lysates were immunoblotted for P23 (A, top left), CHIP (A, top right), and A2A (B, top). G protein β-subunits (Gβ) were visualized as loading control (lower blots in A and B). C, immunoblots done as in B (top) were analyzed with ImageJ. The ratio of mature (M) and core (C) glycosylated species was determined and normalized by setting the mean ratio observed in untreated control cells as 1. Data are means from three independent experiments; error bars, S.D. Statistical significance was assessed by analysis of variance followed by Dunnett's multiple comparison post hoc test. *, p < 0.05.