Figure S5.
ErbB3 enhances Integrin β1 stability and binds to GGA3 and Rabaptin5 in vitro. (A) Immunoblotting analysis of the indicated proteins and GAPDH (as a loading control) in MCF10A cells, transfected with control (siCtrl) or ErbB3 siRNA #2 prior to treatment with protein synthesis inhibitor CHX or vehicle alone for the indicated times in hours. (B) Quantification of Integrin β1 band intensities presented as mean values ± SEM; n = 3 independent experiments. (C) Protein expression of ErbB3, Integrin β1, and the indicated EV-specific positive (ALIX and TSG101) and α-Tubulin (as a loading control) in the cell extract of MCF10A control or ErbB3-depleted samples. (D) Confocal imaging of colocalization of endogenous ErbB3 with ectopically expressed mCherry-GGA3 and endogenous Rabaptin5 in MCF7 cells. (E) Analysis of ErbB3 colocalization with mCherry-GGA3 or Rabaptin5. The relative ErbB3 enrichment at the positive colocalization was determined by the formula (a−b)/b, where a is the ErbB3 intensity of mCherry-GGA3 or Rabaptin5 structure center, and b is the adjacent volume (background) for each structure. Each data point represents the minimum average of 20 structures in one cell. (F) GGA3 protein–protein interaction network analysis using STRING and a confidence score >0.7. (G) Immunoblotting analysis of MCF10A cells, transfected with control (siCtrl) or ErbB3 siRNA #2 prior to treatment with the proteasome inhibitor MG132 (50 µM) for the indicated times in hours. MG132 partially restores GGA3 and Rabaptin5 levels in ErbB3-depleted cells. (H) In vitro binding of ErbB3 to Rabaptin5. Immunoblotting of ErbB3 immunoprecipitates following incubation of recombinant ErbB3 and Rabaptin5. (I) Rabaptin5, ErbB3, Integrin β1, and β-Actin (as a loading control) protein levels in MCF7 cells transiently transfected with control siRNA (siCtrl) or siRabaptin5. (J) EVs enriched in the VSF released by MCF7 transiently transfected with siCtrl or siRabaptin5 were quantified by NTA in terms of nanoparticle modal size (nm) and number upon normalization to the total cell number. (K) Representative TEM micrographs of EVs enriched in the VSF of control or Rabaptin5-depleted MCF7 cells (EVs are indicated by red arrows). (L) Related to Fig. 6 J. Densitometric quantification of the indicated protein expression in MCF10A cells is presented as the mean ± SEM from three biological replicates, normalized to the corresponding siCtrl condition. Data are presented as mean values ± SEM. The P value in B was determined by two-tailed paired Student’s t test, while in J, the P value was determined by unpaired Student’s t test. P values in L were determined by one-way ANOVA followed by multiple paired comparisons conducted by Bonferroni’s posttest method. *P ≤ 0.05; ****P ≤ 0.0001. Scale bars, 10 µm (B), 500 nm (C).

ErbB3 enhances Integrin β1 stability and binds to GGA3 and Rabaptin5 in vitro. (A) Immunoblotting analysis of the indicated proteins and GAPDH (as a loading control) in MCF10A cells, transfected with control (siCtrl) or ErbB3 siRNA #2 prior to treatment with protein synthesis inhibitor CHX or vehicle alone for the indicated times in hours. (B) Quantification of Integrin β1 band intensities presented as mean values ± SEM; n = 3 independent experiments. (C) Protein expression of ErbB3, Integrin β1, and the indicated EV-specific positive (ALIX and TSG101) and α-Tubulin (as a loading control) in the cell extract of MCF10A control or ErbB3-depleted samples. (D) Confocal imaging of colocalization of endogenous ErbB3 with ectopically expressed mCherry-GGA3 and endogenous Rabaptin5 in MCF7 cells. (E) Analysis of ErbB3 colocalization with mCherry-GGA3 or Rabaptin5. The relative ErbB3 enrichment at the positive colocalization was determined by the formula (a−b)/b, where a is the ErbB3 intensity of mCherry-GGA3 or Rabaptin5 structure center, and b is the adjacent volume (background) for each structure. Each data point represents the minimum average of 20 structures in one cell. (F) GGA3 protein–protein interaction network analysis using STRING and a confidence score >0.7. (G) Immunoblotting analysis of MCF10A cells, transfected with control (siCtrl) or ErbB3 siRNA #2 prior to treatment with the proteasome inhibitor MG132 (50 µM) for the indicated times in hours. MG132 partially restores GGA3 and Rabaptin5 levels in ErbB3-depleted cells. (H) In vitro binding of ErbB3 to Rabaptin5. Immunoblotting of ErbB3 immunoprecipitates following incubation of recombinant ErbB3 and Rabaptin5. (I) Rabaptin5, ErbB3, Integrin β1, and β-Actin (as a loading control) protein levels in MCF7 cells transiently transfected with control siRNA (siCtrl) or siRabaptin5. (J) EVs enriched in the VSF released by MCF7 transiently transfected with siCtrl or siRabaptin5 were quantified by NTA in terms of nanoparticle modal size (nm) and number upon normalization to the total cell number. (K) Representative TEM micrographs of EVs enriched in the VSF of control or Rabaptin5-depleted MCF7 cells (EVs are indicated by red arrows). (L) Related to Fig. 6 J. Densitometric quantification of the indicated protein expression in MCF10A cells is presented as the mean ± SEM from three biological replicates, normalized to the corresponding siCtrl condition. Data are presented as mean values ± SEM. The P value in B was determined by two-tailed paired Student’s t test, while in J, the P value was determined by unpaired Student’s t test. P values in L were determined by one-way ANOVA followed by multiple paired comparisons conducted by Bonferroni’s posttest method. *P ≤ 0.05; ****P ≤ 0.0001. Scale bars, 10 µm (B), 500 nm (C).

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