Dynein trafficking of Kv7.4 is dependent on cholesterol. (A i) Representative images of PLAs in smooth muscle cells from rat mesenteric arteries with Kv7.4 and dynein antibodies in control (left) and M-βCD–treated (right) cells. Red puncta indicate target proteins are in close proximity (<40 nM). Scale bars, 10 μm. (A ii) Quantification of the number of PLA puncta in mesenteric artery myocytes (control, 20 cells, n = 2 rats; M-βCD, 18 cells, n = 2 rats) showing significant decrease of Kv7.4 and caveolin-1 colocalization in M-βCD–treated cells (***, P < 0.0001 according to an unpaired t test). (B i) Representative midcell z section of a mesenteric artery myocyte treated with or without M-βCD and stained for caveolin-1 (Cav-1; magenta) and Kv7.4 or NCX (green). (B ii) Mean membrane intensity of Kv7.4 relative to total intensity in cells increased with M-βCD (n = 10) compared with nontreated control cells (n = 8; according to a one-way ANOVA. ***, P < 0.0001). Scale bars, 5 μm. (B iii) Mean membrane intensity of NCX relative to total intensity in cells was equal in M-βCD–treated cells (n = 10) and nontreated control cells (n = 9–11). The intensity of caveolin-1 was the same for both groups. Error bars show mean values and SEM.