Figure 2.
The C-terminus tail of Rad is necessary for t-tubular expression in adult murine ventricular cardiomyocytes. (A) SR SIM maximum intensity projections of fixed adult murine cardiomyocytes stained with anti-Flag to visualize Flag tagged Rad in two transgenic mice, full-length Flag-Rad, and C-terminus ablated Flag-RadΔCT. A no primary antibody control image is provided using the same acquisition and brightness and contrast settings. Scale bar, 5 μm; magnification is the same for all images. (B) Confocal micrographs of adult murine cardiomyocytes from Flag-Rad or Flag-RadΔCT. As a negative control for Flag immunofluorescence, a cardiomyocyte from a Rad knockout mouse is shown (right panel). Scale bar, 5 μm. Fluorescence intensity profiles of regions of interest from the representative images and their FFT power spectrums. (C) Fundamental peak power of anti-Flag immunostaining that represents a signal with periodicity consistent with t-tubular expression. The FFT peak power median of Flag-Rad 40.9 (IQR: 20.4-87.6; N = 6 mice, n = 41 cells, 93 images) was significantly 476% higher than Flag-RadΔCT 7.1 (IQR: 2.6-20.6; N = 6 mice, n = 41 cells, 92 images). Statistical significance was determined by a linear mixed model, nesting cells into the random factor mouse. Genotype as a main effect was significantly different (P = 0.002, F = 16.502). The estimated marginal means contrast with P value adjustment (Holm) for multiple comparisons (see Fig. 5) between the Flag-Rad and Flag-RadΔCT cells untreated was significantly different (P = 0.001). Boxes are interquartile ranges and whiskers, min to max. 76% of Flag-Rad versus 22% of Flag-RadΔCT cells had FFT power >10. Blinded researchers identified 79% of Flag cells and 7% of Flag-RadΔCT cells as having Flag-staining in an organized t-tubular pattern in at least one region of the cell. Two to four technical replicate images’ FFT power was averaged per cell. (D) A Flag-Rad cardiomyocyte was stained for anti-Flag Rad and anti-CaV1.2. The top 15% gray value intensities are highlighted in cyan in both micrographs. The last micrograph shows pixels that contain both anti-Flag and anti-CaV1.2 staining if that pixel was in the top 15% of gray values in both channels. Scale bar, 5 μm; magnification is the same for all images.

The C-terminus tail of Rad is necessary for t-tubular expression in adult murine ventricular cardiomyocytes. (A) SR SIM maximum intensity projections of fixed adult murine cardiomyocytes stained with anti-Flag to visualize Flag tagged Rad in two transgenic mice, full-length Flag-Rad, and C-terminus ablated Flag-RadΔCT. A no primary antibody control image is provided using the same acquisition and brightness and contrast settings. Scale bar, 5 μm; magnification is the same for all images. (B) Confocal micrographs of adult murine cardiomyocytes from Flag-Rad or Flag-RadΔCT. As a negative control for Flag immunofluorescence, a cardiomyocyte from a Rad knockout mouse is shown (right panel). Scale bar, 5 μm. Fluorescence intensity profiles of regions of interest from the representative images and their FFT power spectrums. (C) Fundamental peak power of anti-Flag immunostaining that represents a signal with periodicity consistent with t-tubular expression. The FFT peak power median of Flag-Rad 40.9 (IQR: 20.4-87.6; N = 6 mice, n = 41 cells, 93 images) was significantly 476% higher than Flag-RadΔCT 7.1 (IQR: 2.6-20.6; N = 6 mice, n = 41 cells, 92 images). Statistical significance was determined by a linear mixed model, nesting cells into the random factor mouse. Genotype as a main effect was significantly different (P = 0.002, F = 16.502). The estimated marginal means contrast with P value adjustment (Holm) for multiple comparisons (see Fig. 5) between the Flag-Rad and Flag-RadΔCT cells untreated was significantly different (P = 0.001). Boxes are interquartile ranges and whiskers, min to max. 76% of Flag-Rad versus 22% of Flag-RadΔCT cells had FFT power >10. Blinded researchers identified 79% of Flag cells and 7% of Flag-RadΔCT cells as having Flag-staining in an organized t-tubular pattern in at least one region of the cell. Two to four technical replicate images’ FFT power was averaged per cell. (D) A Flag-Rad cardiomyocyte was stained for anti-Flag Rad and anti-CaV1.2. The top 15% gray value intensities are highlighted in cyan in both micrographs. The last micrograph shows pixels that contain both anti-Flag and anti-CaV1.2 staining if that pixel was in the top 15% of gray values in both channels. Scale bar, 5 μm; magnification is the same for all images.

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