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Fig. 4 | Molecular Brain

Fig. 4

From: Direct interaction with 14–3-3γ promotes surface expression of Best1 channel in astrocyte

Fig. 4

14–3-3γ -shRNA reduced the expression of 14–3-3γ and membrane expression of Best1 in astrocyte. a 14–3-3γ shRNA reduced the level of 14–3-3γ expression mRNA in cultured astrocytes as determined by Western blot. Cultured astrocytes were transfected with pSicoR-control-shRNA or pSicoR-14-3-3γ-shRNA and β-actin was used as an internal control. b Confocal immunofluorescence images of cultured astrocytes transfected with pSicoR-14-3-3γ-shRNA-mCherry. Scale bar, 50 μm. c Quantification of the two-dimensional cell area by counting the number of GFAP positive pixel for the experiment described in (a). Numbers of determinations are indicated on the bar graph. NS p = 0.5726 (unpaired two-tailed t test). d Representative I–V responses of cultured astrocyte expressing pSicoR-control-shRNA or pSicoR-14-3-3γ-shRNA under whole-cell patch-clamp configuration using 4.5 μM Ca2+-containing or 0 Ca2+-containing patch pipette solution. e Bar graph showing summary of current amplitudes (mean ± SEM). The current responses were recorded in response to a voltage ramp command (from −100 to +100 mV, 1 s duration, 0.2 Hz; Vh of −70 mV). Numbers of determinations are indicated on the bar graph. Asterisk indicates a significant difference determined by one-way ANOVA test and Tukey’s multiple comparison test (****p < 0.0001, ***p = 0.0001, NS p = 0.7227). f Averaged I–V responses of cultured astrocytes expressing Best1-C1 or Best1-C3 under whole-cell patch-clamp configuration using 4.5 μM Ca2+-containing patch pipette solution. g Bar graph showing summary of current amplitudes (mean ± SEM). The current responses were recorded in response to a voltage ramp command (from −100 to +100 mV, 1 s duration, 0.2 Hz; Vh of −70 mV). Numbers of determinations are indicated on the bar graph. Asterisk indicates a significant difference determined by unpaired two-tailed t-test (***p < 0.001). h Cell-surface biotinylation assay in cultured astrocytes. When astrocytes transiently transfected with 14–3-3γ-shRNA, surface expression of Best1 is dramatically decreased. i Cultured astrocytes transfected with pSicoR-control-shRNA-mCherry (upper panel) or pSicoR-14-3-3γ-shRNA-mCherry (bottom panel) were imaged by confocal microscopy using antibodies against Best1 and 3D reconstructions were generated from stacks of images. Scale bars, 20 μm

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