Confocal micrographs of fixed adult murine ventricular cardiomyocytes immunostained with anti-Flag antibody to detect Flag-Rad localization. (A) Cells were β-adrenergic receptor agonist treated isolated from transgenic mice with full-length Flag-Rad (N = 3 mice, n = 8 cells displayed) or Flag-RadΔCT (N = 3 mice, n = 8 cells displayed), representative of a total of 6 and 6 mice, 41 and 41 cells, and 93 and 92 images. Note that the bottom right Flag-RadΔCT image showing t-tubular expression is from the same cell in Fig. 2, representative of the observation that 7% of Flag-RadΔCT cells show t-tubular like expression in at least one region, but not regularly throughout the entire cell (0%). Scale bar, 5 µm; magnification is the same for all images. (B) Confocal microscopy controls were imaged and stained with the same protocols and with the same visualization background and contrast adjustments. For controls of the anti-Flag Rad antibody, a cardiomyocyte from a Rad knockout mouse (Rad−/−) was imaged along with a Flag-Rad cardiomyocyte incubated with no primary antibody imaged with the same settings used for anti-Flag-Rad acquisition. Additionally, a Flag-Rad cardiomyocyte was incubated with no secondary antibody (no fluorophore) and imaged. Lastly, as a control for the commercial CaV1.2 antibody (image presented in Fig. 2 C), a no primary anti-CaV1.2 antibody incubated Flag-Rad cardiomyocyte was imaged the same settings used for CaV1.2 imaging.
