EHD2 is SUMOylated by SUMO2/3 upon mechanical stress. (A) Representative wide-field fluorescence (left) and quantification (right) of in situ PLA experiments in Hs578T cells monitoring EHD2 and SUMO2/3 interaction in the whole cell (Cell), the nucleus (Nucl), and the cell minus the nucleus (Cyto + plasma membrane) under resting (Iso, n = 51), under hypo-osmotic (Hypo, n = 51), and after return to iso-osmotic (Rec, n = 50) conditions. (B) Representative z-projection (average intensity) of a confocal stack of a PLA experiment monitoring EHD2 and SUMO2/3 interaction (red signal) in MLEC cells 5 min after 30 mOsm hypo-osmotic shock. A confocal z cross section along the dashed line shows localization of PLA spots in the nucleus (DAPI; gray). (C) Immunoblot analysis (left) and quantification (right; SUMO2/3 level normalized to GFP) of EGFP-EHD2 SUMOylation by SUMO2/3 in immunoprecipitates from stable HeLa His-SUMO2/3 cells transfected with EHD2-EGFP or EGFP under Iso and Hypo conditions. (D) Same PLA experiments as in A performed in MLEC WT (Iso, n = 76; Hypo, n = 77; Rec, n = 72) or Cav1^−/− cells (Iso, n = 75; Hypo, n = 75; Rec, n = 75). Scale bar = 10 µm; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; in A and D, Dunnett’s multiple comparison test, data are representative of three experiments, mean ± SEM; in C, repeated measures one-way ANOVA; data are mean ± SEM.