FIGURE 8.
Orai1 and CaV1.2 co-localization in aortic myocytes. A, representative images of aortic VSMC using primary antibodies against CaV1.2 and Orai1 conjugated with the appropriate PLA probes, from untreated control cells or from cells stimulated with 5-HT (10 μm), TG (2 μm), or high KCl (70K, 70 mm). Red puncta indicate that proteins are in close proximity (<40 nm). The left panels show VSMC images when antibodies were conjugated with PLA probes, the middle panels show merges images from cells also stained with DAPI (blue), and the right panels show bright field (BF) pictures. B, images from VSMC using primary antibodies against α-SMA and vimentin used as positive control (upper panel) and from VSMC conjugated with only anti-Orai1 primary antibody as negative control (lower panel). C, the bar graph summarizes the mean number of PLA signals in untreated control VSMC (15.42% ± 1.50; n = 6), and in VSMC stimulated with 5-HT (21.60% ± 1.65; n = 10), TG (28.90% ± 3.81; n = 8), or 70K (18.33% ± 2.45; n = 8). No detectable PLA puncta were observed in control experiments using cells conjugated only with anti-Orai1 antibody (Orai1, n = 8). The interaction between anti-α-SMA and anti-vimentin antibodies was used as positive control (100% ± 25.88; n = 5). Similar experiments were repeated 3 times. Values are the percentage of mean ± S.E., normalized to positive control. Significance values indicate difference as compared with control, *, p < 0.05.