Fig. 4. Endophilin A2 co-localizes with the TCR upon activation and regulates TCR internalization and signaling.

a EA2 co-localization with the TCR in Jurkat cells after anti-CD3/CD28 stimulation determined by proximity-ligase assay and visualized as TexasRed⁺ spots using confocal imaging. b Percentage of internalized TCR in T cells from 5 DA and 5 DA^Mut rats after anti-CD3/CD28 stimulation. Data has been reproduced two times. c Percentage of internalized TCR in CD4⁺CD3⁺ T cells from three SH3gl1^−/− and SH3gl1^+/+ mice stimulated with anti-CD3/CD28 for 30 min in the presence or absence of Brefeldin A at 1.25 μg/ml. Experiment has been repeated twice with the same results. d Representative Western blot analysis from two experiments of phosphorylated and unphosphorylated ZAP70 and ERK1/2 in T cells from two SH3gl1^−/− and SH3gl1^+/+ mice stimulated with anti-CD3/CD28 for 0, 3, and 7 min. Histone 2B was used as loading control. e Normalized OD values of phosphorylated Zap70 compared to unphosphorylated Zap70 from two SH3gl1^−/− and SH3gl1^+/+ mice. f Normalized OD values of phosphorylated ERK1/2 compared to unphosphorylated ERK1/2 from two SH3gl1^−/− and SH3gl1^+/+ mice. g Flow cytometry blot showing in vitro cell proliferation of DA^Mut and DA T cells 72 h after anti-CD3/CD28 stimulation. h Division index of sorted T cells from 7 DA and 5 DA^Mut rats after 72 h of anti-CD3/CD28 stimulation. Non-parametrical Mann–Whitney U test was used for statistical evaluation of data. Data are presented as mean with error bars indicating ± SEM with each dot representing an individual value.