Figure 4: Thr9 in segment a of ANO1 is required for binding to 14-3-3γ. | Scientific Reports

Figure 4: Thr9 in segment a of ANO1 is required for binding to 14-3-3γ.

From: Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells

Figure 4

(a) Alignment of amino acid sequence of 14-3-3 binding motif in human, mouse, rat, and bovine ANO1. The red arrow indicates a predicted residue for phosphorylation-dependent 14-3-3 binding (http://scansite.mit.edu/motifscan_seq.phtml). (b) MBP pull-down assay showed that seg(a)-T9A bound to 14-3-3γ more poorly than seg(a) WT. (c) Cell surface biotinylation results from membrane protein fractions from HEK293T cells transfected with GFP-ANO1 WT or GFP-ANO1-T9A. Western blot images (b,c) were cropped for clarification. Full blot images were included in supplementary information. (d) Normalized bar graph showing data obtained from three independent experiments as in (c). Data are expressed as mean ± SEM; **P < 0.01. (e) Representative traces of whole-cell recordings in COS7 cells transfected with GFP-ANO1, GFP-ANO1-T9A, and/or mCh-14-3-3γ. (f) Summary bar graph showing normalized current density as in (g) at +80 mV. Data are expressed as mean ± SEM; n.s. = not significant, *P < 0.05, **P < 0.01.

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