Fig. 5.

Characterization of the role and distribution of SAP97 on ISO-mediated translocation of WT β₁-AR in ARVMs. (A) Western blotting of cell lysates (50 µg) prepared from ARVMs that were infected with scrambled Ad-shRNA or Ad-SAP97 shRNA, and then probed with anti-SAP97 IgG. (B) Effect of SAP97 knockdown on WT β₁-AR compartmentalization in ARVMs. ARVMs infected simultaneously with 50 multiplicities of infection (m.o.i.) of Ad-WT FLAG β₁-AR and with either 50 m.o.i. scrambled Ad-shRNA (images a–c) or Ad-SAP97 shRNA (images d–f) were used. These slides were incubated with buffer (images a and d), ISO for 30 minutes (images b and e) or ISO/ALP (images c and f) and processed as described in Fig. 1A. Each scale bar in (B) represents 15 µm. (C) Effect of scrambled vs. SAP97 shRNA on the mean ± S.E. for the distribution of WT β₁-AR pixels outside the 400-nm partition in ARVMs exposed to buffer, ISO, and ISO/ALP is presented. These data were derived from 10 images/condition for three separate experiments, n = 30 images). Statistical comparisons were carried out by one-way ANOVA followed by Bonferroni’s test. NS, indicates nonsignificant differences between the column pairs or *, **, and *** to indicate P < 0.05, P < 0.01, and P < 0.001, respectively. (D) Dual-fluorescence confocal microscopy between WT β₁-AR (pseudo red) and SAP97 (pseudo green) in ISO-exposed ARVMs. (E) Statistical comparisons in the percentile of overlapping pixels between the two groups were carried out by student’s t test. P values are expressed as NS to indicate no significant difference or *, **, and *** to indicate P < 0.05, P < 0.01, and P < 0.001, respectively.