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. 2013 Mar 25;288(20):14018–14031. doi: 10.1074/jbc.M113.454439

FIGURE 4.

FIGURE 4.

Clathrin and ankyrin-G cooperate in localization of E-cadherin to MDCK cell lateral membranes. A, left, shown is a schematic representation of E-cadherin. Cadherin repeats (light blue), transmembrane segment (gray), ankyrin-G-binding site (yellow), p120 site (red), and β catenin site (green). Ankyrin-G binding sequences are shown below for wild-type (top), Poly(A) (2nd row), LL-AA (3rd row), and Poly(A) + LL-AA mutant (bottom) E-cadherin. Residues critical for ankyrin-G binding are shown in red, and the dileucine motif is highlighted in yellow. Right, membrane recruitment of ankyrin-G in HEK293 cells is shown. Cells were either transfected with ankyrin-G alone (control, white) or plus Wild-type (black), Poly(A) (blue), LL-AA (green), or Poly(A) + LL-AA (red) E-cadherin. Line fluorescence intensity analysis was performed on a single plane, and peak pixel intensity on the membrane was compared with average pixel intensity in cytoplasm. *, p < 0.05 compared with control, Poly(A), and Poly(A) + LL-AA mutant (one way ANOVA followed by the Tukey post hoc test, n = 10 cells for each condition). B, MDCK cells stably expressing an inducible clathrin shRNA were transfected with wild-type (top), Poly(A) (2nd row), LL-AA (3rd row), or Poly(A) + LL-AA (bottom) E-cadherin in the absence (+clathrin (+CHC)) or presence (−clathrin) of doxycycline induction of clathrin shRNA expression. XZ projections shown on the left. The bar represents 10 μm. XY projection of apical planes are shown on the right. C, shown is quantification of the apical mean pixel intensity in the absence (+clathrin) or presence (−clathrin) of doxycycline induction of clathrin shRNA expression. Mean pixel intensity of a three-dimensional region of interest containing the apical membrane was quantified and compared with a three-dimensional region of interest containing the lateral membrane. *, p < 0.05 compared with WT + clathrin. #, p < 0.05 compared with Poly(A) +clathrin (one-way ANOVA followed by the Tukey post hoc test, n = 5–8 cells for each condition). D, shown is a Western blot of MDCK cells stably expressing either luciferase shRNA (Luc, left) or clathrin shRNA (right) demonstrating silencing of clathrin (top) with GAPDH used as a loading control (bottom).