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. 2013 Sep 3;288(41):29453–29466. doi: 10.1074/jbc.M113.468801

FIGURE 3.

FIGURE 3.

GAP activity of SNX26 is required to decrease the F-actin content in COS-7 cells as well as in dendritic spines of neurons. A, COS-7 cells expressing GFP, GFP-SNX26, GFP-RhoGAP, GFP-RI-RhoGAP, or GFP-R350I were fixed and stained with TxRed-phalloidin to visualize F-actin. Scale bar, 10 μm. B, normalized intensity ratio (transfected/untransfected (Tf/unTf)) of cells expressing SNX26 or RhoGAP was significantly lower than that of cells expressing GFP (CTL, control), GFP-RI-RhoGAP (GAP activity-deficient mutant of RhoGAP domain), or R350I (GAP activity-deficient mutant of SNX26 FL). Data are presented as means ± S.E. of three independent experiments in which >40 transfected cells were examined for each group. ***, p < 0.001 (ANOVA and Tukey's HSD post hoc test). C, representative images of dendritic spines of neurons. Cultured hippocampal neurons were transfected at DIV 15 with cyan fluorescent protein and with GFP or GFP-SNX26 FL, fixed at DIV 16, and then stained with TxRed-phalloidin. Scale bar, 2 μm. D, TxRed intensities in dendritic spines and neighboring dendritic shafts were compared. Over 30 transfected individual spines and dendrites were examined for each group. Data are presented as means ± S.E. of three independent experiments. The normalized intensity ratio (spine/shaft) of cells expressing SNX26 FL was much lower than that of cells expressing control (CTL). ***, p < 0.001 (ANOVA and Tukey's HSD post hoc test). E, representative images of dendritic spines of neurons. Cultured hippocampal neurons were transfected at DIV 15 with GFP, GFP-RhoGAP, or GFP-RI-RhoGAP, fixed at DIV 16, and then stained with TxRed-phalloidin. Scale bar, 2 μm. F, TxRed intensities in dendritic spines and neighboring dendritic shafts were compared. Over 30 transfected individual spines and dendrites were examined for each group. Data are presented as means ± S.E. of three independent experiments. The normalized intensity ratio (spine/shaft) of cells expressing RhoGAP was much lower than that of cells expressing control or RI-RhoGAP. ***, p < 0.001 (ANOVA and Tukey's HSD post hoc test).