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. 2015 Nov 17;291(3):1076–1091. doi: 10.1074/jbc.M115.676882

FIGURE 4.

FIGURE 4.

Membrane-binding domain mutants fail to rescue CARMIL2-depletion phenotypes. A, immunoblot showing depletion of endogenous CARMIL2 and expression of CARMIL2-GFP mutants. B, representative images reveal shapes of single migrating cells. Initial frames from 1-h movies (supplemental Movies S11–S17). Scale bar, 10 μm. C, quantitation of polarity of cells from images similar to those in panel B. Circularity of cell border calculated as described under “Experimental Procedures.” Box and whisker plots indicate the median, interquartile range, and 10th and 90th percentiles. Statistical analysis: n = 30 cells per sample, double asterisk, indicates p < 0.0001, and single asterisk, indicates p < 0.001. Values are not significantly different between CARMIL2-shRNA and mutant-rescue cells. D, macropinosomes in CARMIL2-depleted and rescue expression cells, counted in initial frames of movies (n = 30 cells). Plotted values are mean ± S.E. Asterisks indicate p < 0.0001. Values are not significantly different between CARMIL2-shRNA and mutant rescue cells. E, leading edges of CARMIL2-depleted and rescue expression cells stained with anti-vimentin, fluorescent phalloidin, or anti-CP. Arrowheads indicate F-actin and arrows indicate CP in lamellipodia. Scale bar, 10 μm.