Figure S2.
ErbB3 promotes endocytic recycling of Integrin β1 in the absence of EGFR or ErbB2. (A) Immunoblotting analysis of the indicated proteins and α-Tubulin (as a loading control) in the total MCF10A cell lysates under GF-deprived culture conditions or upon supplementation of the EGF and βNRG, after depletion of ErbB3, EGFR, or ErbB2. (B) Immunoblotting analysis of ErbB3, Integrin β1, and GAPDH (as a loading control) upon transfection of prHMEC cells with control or ErbB3 siRNA#1. (C) Relative levels of Integrin β1 (ITGB1) mRNA in MCF10A control or ErbB3 siRNA#1-transfected cells as determined by RT-qPCR (n = 6 independent experiments). Data presented in C are shown as the mean ± SEM. The P value was determined by the Mann–Whitney U test. (D) Immunofluorescence imaging of the surface pool of Integrin β1 labeled with Alexa 488–conjugated antibody on ice for 1 h, on MCF10A cells transfected with control siRNA (siCtrl) or siRNAs targeting EGFR or ErbB2. (E and F) Quantification of Integrin β1 fluorescence intensity upon silencing of EGFR (E) or ErbB2 (F) and normalized against the respective control siRNA-treated samples. Data are presented as mean values ± SEM. (G) Live-cell TIRF imaging of MCF10A cells to monitor recycling of labeled Integrin β1, after prior siRNA-mediated depletion of ErbB2, EGFR, or ErbB3 (#1 and #2). (H) Related to Fig. 2, J and K: shows AUC of the Integrin β1 recycling data. Data are presented as mean values ± SEM. n values are indicated in main figures. (I and J) Expression of siRNA-resistant ErbB3 restores the surface pool of traced Integrin β1 in ErbB3-depleted MCF10A cells. (I) Confocal imaging of Integrin β1 labeled on the cell surface with an Alexa 488–conjugated antibody, prior to (0 h) or after tracing (1 h), on cells expressing siRNA-resistant mCh-ErbB3 or fluorophore alone with or without transfection of control (siCtrl) or ErbB3 siRNA as indicated. (J) Fluorescence intensity (Integrin β1) along cell–cell borders was quantified from three independent experiments. Values were normalized against intensities prior to tracing (0 h). The results suggest that off-target effects of the ErbB3 siRNAs do not underlie the observed recycling defect. Data are presented as mean values ± SD; n > 230 cell–cell borders from 3 independent experiments, and P values were determined by two-tailed paired Student’s t test. (K) Immunoblotting analysis of ErbB3 immunoprecipitates or total cell lysates of MCF10A cells cultured in the presence of 5% horse serum or under GF-deprived culture conditions. Antibodies detecting total phosphotyrosine (4G10; pTyr) or the specific phosphotyrosine 1289 on ErbB3 were used, and α-Tubulin was used as a loading control for the total cell lysates. Scale bars, 20 µm (D and G), 10 µm (I). GF, growth factor.

ErbB3 promotes endocytic recycling of Integrin β1 in the absence of EGFR or ErbB2. (A) Immunoblotting analysis of the indicated proteins and α-Tubulin (as a loading control) in the total MCF10A cell lysates under GF-deprived culture conditions or upon supplementation of the EGF and βNRG, after depletion of ErbB3, EGFR, or ErbB2. (B) Immunoblotting analysis of ErbB3, Integrin β1, and GAPDH (as a loading control) upon transfection of prHMEC cells with control or ErbB3 siRNA#1. (C) Relative levels of Integrin β1 (ITGB1) mRNA in MCF10A control or ErbB3 siRNA#1-transfected cells as determined by RT-qPCR (n = 6 independent experiments). Data presented in C are shown as the mean ± SEM. The P value was determined by the Mann–Whitney U test. (D) Immunofluorescence imaging of the surface pool of Integrin β1 labeled with Alexa 488–conjugated antibody on ice for 1 h, on MCF10A cells transfected with control siRNA (siCtrl) or siRNAs targeting EGFR or ErbB2. (E and F) Quantification of Integrin β1 fluorescence intensity upon silencing of EGFR (E) or ErbB2 (F) and normalized against the respective control siRNA-treated samples. Data are presented as mean values ± SEM. (G) Live-cell TIRF imaging of MCF10A cells to monitor recycling of labeled Integrin β1, after prior siRNA-mediated depletion of ErbB2, EGFR, or ErbB3 (#1 and #2). (H) Related to Fig. 2, J and K: shows AUC of the Integrin β1 recycling data. Data are presented as mean values ± SEM. n values are indicated in main figures. (I and J) Expression of siRNA-resistant ErbB3 restores the surface pool of traced Integrin β1 in ErbB3-depleted MCF10A cells. (I) Confocal imaging of Integrin β1 labeled on the cell surface with an Alexa 488–conjugated antibody, prior to (0 h) or after tracing (1 h), on cells expressing siRNA-resistant mCh-ErbB3 or fluorophore alone with or without transfection of control (siCtrl) or ErbB3 siRNA as indicated. (J) Fluorescence intensity (Integrin β1) along cell–cell borders was quantified from three independent experiments. Values were normalized against intensities prior to tracing (0 h). The results suggest that off-target effects of the ErbB3 siRNAs do not underlie the observed recycling defect. Data are presented as mean values ± SD; n > 230 cell–cell borders from 3 independent experiments, and P values were determined by two-tailed paired Student’s t test. (K) Immunoblotting analysis of ErbB3 immunoprecipitates or total cell lysates of MCF10A cells cultured in the presence of 5% horse serum or under GF-deprived culture conditions. Antibodies detecting total phosphotyrosine (4G10; pTyr) or the specific phosphotyrosine 1289 on ErbB3 were used, and α-Tubulin was used as a loading control for the total cell lysates. Scale bars, 20 µm (D and G), 10 µm (I). GF, growth factor.

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