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. 2012 Nov 5;287(52):43995–44005. doi: 10.1074/jbc.M112.417501

FIGURE 4.

FIGURE 4.

C70A mutation prevents AnkG retention in MDCK cells grown in low calcium. A, stable MDCK cells expressing WT or C70A AnkG-GFP were grown in low calcium medium overnight and then fixed and immunostained against GFP. B, stable MDCK cells expressing WT or C70A AnkG-GFP were grown in normal medium to reach a polarized monolayer and then fixed and immunostained against GFP. C, 60 ng of WT, C70A, DAR999AAA (DAR), and DAR999AAA/C70A double mutant (C70A, DAR) AnkG-GFP cDNA were transfected into MDCK cells in 14-mm MatTek plates. Cells were then fixed and immunostained against GFP 24 h post-transfection (green channel in grayscale). Scale bars, 10 μm for A–C. D, quantitative results of the fluorescence intensity ratio of plasma membrane versus cytoplasmic staining in AnkG stable MDCK cell lines. Data were compared using one-way ANOVA followed by a Tukey post hoc test (p < 0.001 for ANOVA, n = 26–31 from two independent experiments). *, p < 0.05; ns, not significant. E, quantitative results of the fluorescence intensity ratio of plasma membrane versus cytoplasmic staining of WT, C70A, DAR999AAA (DAR), and DAR999AAA/C70A double mutant (C70A, DAR) transfected into MDCK cells. Data were compared using one-way ANOVA followed by a Tukey post hoc test (p < 0.001 for ANOVA, n = 26–28 from three independent experiments). *, p < 0.05. Error bars, S.E.