Fig. 5
Erbin is required for interactions between HER2/NHERF1/Ezrin in membrane protrusions. A-C) PLA fluorescence (red) showing protein interaction between HER2/NHERF1 (A), HER2/Ezrin (B), and NHERF1/Ezrin (C) in MCF10A, SKBR3, and Erbin knockdown SKBR3 cells. Top of cells; focused on surface of plasma membrane, Middle of cells; focused on the mid-portion of cells to examine intracellular interactions. Phalloidin staining in green outlines the sub-cortical actin beneath the plasma membrane. All scale bars represent 10µM. Bar graphs show quantification of PLA results by measuring intensity of fluorescence from PLA signal at the surface and inside of cells (n = 3). HER2/NHERF1 (control n = 16, ErbinKD n = 10), HER2/Ezrin (control n = 14, ErbinKD n = 14), NHERF1/Ezrin (control n = 14, ErbinKD n = 14). Bars represent the mean ± SEM. D-F) PLA showing protein interaction of HER2/Erbin (D), Erbin/NHERF1 (E), and Erbin/Ezrin (F) in serum free (SF) and EGF treated SKBR3 cells. Phalloidin staining is in green. All scale bars represent 10µM. Bar graphs show quantification of PLA results by measuring intensity of fluorescence from PLA signal at the surface and inside of cells (n = 3). Erbin/HER2 (control n = 19, ErbinKD n = 19), Erbin/NHERF1 (control n = 19, ErbinKD n = 19), Erbin/EZRIN (control n = 19, ErbinKD n = 19). Bars represent the mean ± SEM. G-I) PLA showing protein interaction between HER2/NHERF1 (G), HER2/Ezrin (H), and NHERF1/Ezrin (I) in control and Erbin knockdown SKBR3 cells under either serum free or serum free with 100ng/ml EGF. All scale bars represent 10µM. Bar graphs show quantification of PLA results by measuring intensity of fluorescence from PLA signal at the surface and inside of cells (n = 3). HER2/NHERF1 (serum free; control n = 20, ErbinKD n = 20, EGF; control n = 13, ErbinKD n = 13), HER2/EZRIN (serum free; control n = 13, ErbinKD n = 13, EGF; control n = 10, ErbinKD n = 10), EZRIN/NHERF1 (serum free; control n = 14, ErbinKD n = 14, EGF; control n = 11, ErbinKD n = 11). Bars represent the mean ± SEM. J) Western blot analysis of HER2 and phospho-HER2 in control and Erbin knockdown SKBR3 cells under serum free and serum free with 100ng/ml EGF. (n = 3) Bar graphs represent the mean ± SEM. *** denotes p < 0.0005, **** denotes p < 0.00005