Fig. 4

The loss of Erbin inhibits HER2 signaling and the formation of membrane protrusions. A) Erbin mRNA expression assessed by QPCR in control and Erbin knockdown SKBR3 cells. (n = 3). B) Western blot analysis of HER2, phospho-HER2, EGFR, and phospho-EGFR in control and Erbin knockdown SKBR3 cells. (n = 3) C) Immunofluorescence for HER2 with phalloidin (top row), NHERF1 (middle row), and Ezrin (bottom row) in Erbin knock down SKBR3 cells. All scale bars represent 10µM. D-F) Scanning electron microscopy images from MCF10A (D), SKBR3 (E), and Erbin knockdown SKBR3 (F) cells. G) Percentage of cells forming membrane protrusions in control and Erbin knockdown SKBR3 cells as assessed by immunofluorescence. Each data point represents the percentage of cells in 8 separate, randomly chosen microscope fields. n = 8 H) Co-immunoprecipitation of HSP90 and HER2. Extracts from control SKBR3 and Erbin knock down SKBR3 cells. Cells were immunoprecipitated with HSP90 antibody and blotted for HSP90 and HER2. I) PLA (red fluorescence) showing protein interaction between HER2/HSP90, Erbin/HSP90, NHERF1/HSP90, and Ezrin/HSP90 in control and Erbin knock down SKBR3 cells. Phalloidin staining in green outlines the sub-cortical actin beneath the plasma membrane. All scale bars represent 10µM. (n = 3) J) Quantification of PLA results (Fig. 3H) as assessed by measuring the intensity of PLA signal fluorescence. HER2/HSP90 (control n = 20, ErbinKD n = 20), Erbin/HSP90 (control n = 20, ErbinKD n = 20), NHERF1/HSP90 (control n = 20, ErbinKD n = 20), EZRIN/HSP90 (control n = 20, ErbinKD n = 20) (n = 3). Bar graphs represent the mean ± SEM. **** denotes p < 0.00005