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. 2016 Sep 13;291(51):26352–26363. doi: 10.1074/jbc.M116.727990

FIGURE 6.

FIGURE 6.

Knockdown of Hap1 reduces the surface level of Cav1.2. A, INS-1 cell overexpression of YH-Cav1.2 together with scramble-siRNA (upper) or Hap1-siRNA (bottom). Profiles of YFP intensity along the yellow lines through the cell bodies are shown at bottom left of the 1st image. The signal of YFP in control cells shows sharp peaks at the edges of the cell membrane, whereas the other traces remain elevated throughout the cell compared with Hap1-siRNA cells. Scale bar, 10 μm. B, INS-1 cells treated with Hap1-siRNA displayed a significant reduction in the level of YH-Cav1.2 surface YFP intensity and average ratios (PM/C) compared with scramble-siRNA control (**, p < 0.001; scramble n = 45, siRNA n = 47). au is arbitrary unit. C, immunofluorescent Cav1.2 staining of INS-1 cells transfected with Hap1-siRNA (bottom) or scramble-siRNA (upper). Line scans of the anti-Cav1.2 fluorescence intensity through the cell bodies are shown in the insets. The signal of YFP in scramble-siRNA cells shows sharp peaks at the edges of the cell membrane, whereas no significant peaks were seen in Hap1-siRNA cells. Scale bar, 10 μm. D, surface Cav1.2 intensity and average ratios (PM/C) of endogenous Cav1.2 surface expression in Hap1-siRNA or scramble-siRNA cells (*, p < 0.01; scramble n = 15, siRNA n = 18). E, immunofluorescent of INS-1 cells with an anti-HA antibody without permeabilization. F, cells treated with Hap1-siRNA displayed a significant reduction in the level of YH-Cav1.2 surface HA intensity compared with scramble-siRNA (upper panel) (**, p < 0.001; scramble n = 195, siRNA n = 147). G, immunoblotting of Cav1.2 in plasma membrane (PM), cytosol (C), and homogenate (H) fractions.