Fig. 3

Erbin is physically associated with the HER2/NHERF1/Ezrin protein complex. A) Immunofluorescence for HER2 and Erbin in day 10 lactating normal mammary gland (top) and day 10 lactating MMTV-Neu mammary gland (bottom). All scale bars represent 10µM. B-C) Immunofluorescence for Erbin with HER2 (top row), NHERF1 (middle row), and Ezrin (bottom row) in hyperplastic lesions in MMTV-Neu mammary glands (B) and human DCIS (C). All scale bars represent 10µM. D-E) Immunofluorescence for Erbin with HER2 (top row), Erbin with NHERF1 (middle row), and Erbin with Ezrin (bottom row) in SKBR3 cells (D) and MCF10A cells (E). All scale bars represent 10µM. F) Co-immunoprecipitation of HER2, Ezrin and Erbin. Extracts from SKBR3 cells were immunoprecipitated with either anti-HER2 antibody or control IgG, and blotted for HER2, Ezrin, and Erbin. G) Co-immunoprecipitation of NHERF1 and Erbin. Extracts from HA-NHERF1 over-expressing SKBR3, MCF10A, and HER2 knock-down SKBR3 cells were immunoprecipitated with either anti-HA antibody or control IgG and blotted for HA and Erbin. H) Proximity ligation assay (PLA) showing protein interactions between HER2/Erbin, Erbin/NHERF1, and Erbin/Ezrin in MCF10A and SKBR3 cells. Phalloidin staining indicates sub-cortical actin. All scale bars represent 10µM. Bar graphs show the quantification of PLA-associated fluorescence at the plasma membrane. HER2/Erbin (MCF10A n = 15, SKBR3 n = 15), Erbin/EZRIN (MCF10A n = 16, SKBR3 n = 16), Erbin/NHERF1 (MCF10A n = 17, SKBR3 n = 17). Bar graphs represent the mean ± SEM. **** denotes p < 0.00005