Rabaptin5 and GGA3 are effectors of ErbB3 in endosomal trafficking. (A and B) Representative TIRF microscopy images of Integrin β1 from peripheral areas of MCF10A cells transiently transfected with control siRNA (siCtrl) or siRabaptin5. (C) Quantification of recycled Integrin β1 was conducted on the indicated number of cells (right-hand side of graphs) from three independent experiments and is shown as Alexa 488 intensity normalized against levels at the onset of tracing (0) and maximum intensity (1). (D) Columns show AUCs. (E) Determination of Integrin β1 turnover by pulse-chase metabolic labeling. Control or Rabaptin5 siRNA-transfected MCF10A cells were pulse chase–labeled with radioactive (³⁵S) methionine and cysteine. Radiolabeled Integrin β1 was visualized by radiography of Integrin β1 immunoprecipitates (upper panel). Cell lysates and immunoprecipitates were analyzed by immunoblotting as indicated. (F) Quantification of radiolabeled Integrin β1 at the indicated times of three independent pulse and chase experiments. Data are presented as mean values ± SEM. P values were determined by two-tailed paired Student’s t test. ns, nonsignificant. (G) Expression of the indicated proteins and GAPDH (as a loading control) in the cell extract of MCF10A cells transiently transfected with siCtrl, siErbB3#2, or siRabaptin5. 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. P values were determined by one-way ANOVA, followed by multiple paired comparisons conducted by means of Bonferroni’s posttest method. *P ≤ 0.05; **P ≤ 0.01. (H) EVs enriched in the VSF released by the indicated MCF10A transiently transfected with control or Rabaptin5 siRNAs were quantified by NTA in terms of nanoparticle modal size (nm) and number after normalization to the total cell number. (I) Representative TEM micrographs of EVs enriched in the VSF of control or Rabaptin5-depleted MCF10A cells (EVs are indicated by red arrows). (J) Protein expression in the cell extract of MCF10A control samples, ErbB3#2- or GGA3-depleted (left panel) samples, of ErbB3, Integrin β1, and GGA3, besides the indicated EV-specific positive markers (ALIX, TSG101, and CD81) and GAPDH (as a loading control). (K) EVs enriched by UC and released by the indicated MCF10A transiently transfected with control or GGA3 siRNAs were quantified by NTA in terms of nanoparticle modal size (nm) and number after normalization to the total cell number. Data in H and K are presented as mean values ± SEM. P values were determined by unpaired Student’s t test. *P ≤ 0.05. (L) Protein expression levels of Integrin β1 and the indicated positive and negative EV markers in the UC EVs isolated from MCF10A cells described in I. Average densitometric values of at least two independent biological replicates were normalized to siRNA control. (M) Representative TEM micrographs of EVs enriched by UC secreted from control, and ErbB3- or GGA3-depleted MCF10A cells (EVs are indicated by red arrows). Scale bars, 10 µm (B), 200 nm (I), 1 µm (M).