Fig. 3: Crbn modulates SOCE mediated by CRAC channels.

a, b BMDMs from the indicated mice a or Crbn-overexpressing LR73 cells b were stained with Fura2-AM and then treated with 1 µM thapsigargin for the indicated duration. Thereafter, 2 mM calcium was added to the cells at the indicated time. Fluorescence of the cells was measured with a microplate reader. AUC area under curve. n = 3 experiments, mean ± SEM. NS not significant (two-tailed unpaired Student t test). c BMDMs derived from WT or Crbn^−/− mice were pre-incubated with 10 µM YM-58483 and then SOCE was measured as in a. d, e BMDMs derived from WT or Crbn^−/− mice were incubated with TAMRA-stained apoptotic cells in 5 mM EGTA d or 10 μM YM-58483 e. Thereafter, engulfing phagocytes were analyzed by flow cytometry. n = 3 experiments, mean ± SEM (two-tailed unpaired Student t test). f, g BMDMs derived from WT and Crbn^−/− mice were incubated with Cy3-labeled PS beads and observed using time-lapse confocal microscopy f. Time required for phagocytic cup closure was measured g. Arrows with a same color indicate a same PS bead being engulfed. Scale bar, 10 µm. n = 3 experiments (each dot represents one PS bead), mean ± SEM. NS not significant (two-way ANOVA). h BMDMs from indicated mice were incubated with TAMRA-stained apoptotic cells in the indicated concentration of Mdivi-1, and engulfing phagocytes were analyzed by flow cytometry. n = 4 experiments, mean ± SEM. NS not significant (one-way ANOVA).