Fig. 6

Ezrin and NHERF1 are required for Erbin and HER2 interactions. A) Immunofluorescence for Erbin with HER2 (top row), NHERF1 (middle row), and Ezrin (bottom row) in NSC668394 treated SKBR3 cells. All scale bars represent 10µM. B) Immunofluorescence for Erbin with HER2 (top row) and Ezrin (bottom row) in NHERF1 knockdown SKBR3 cells. All scale bars represent 10µM. C) PLA fluorescence showing protein-protein interactions between HER2/Erbin, Erbin/NHERF1, or Erbin/Ezrin in control, NSC668394 treated-, and NHERF1 knock-down SKBR3 cells. All scale bars represent 10µM. (n = 3) D) Quantification of PLA results was performed by measuring the fluorescent intensity of PLA signals at the cell surface. HER2/Erbin (control n = 14, NSC668394 n = 14, NHERF1KD n = 14), Erbin/NHERF1 (control n = 19, NSC668394 n = 19, NHERF1KD n = 19), Erbin/EZRIN (control n = 15, NSC668394 n = 15, NHERF1KD n = 15). Bar graphs represent the mean ± SEM. **** denotes p < 0.00005. (n = 3). E) A working model proposing how HER2 mediated disruption of apical/basal polarity creates the opportunity for the formation of a multi-protein signaling complex that includes HER2, Erbin, PMCA2, NHERF1, and Ezrin. Created with BioRender.com