Nfatc2

D1 cells were seeded into 8 well μ-slides (Ibidi) and rested for 2 h before stimulation with curdlan (10 μg/ml) for various times prior to fixation in 2% paraformaldehyde (PFA). The cells were then re-suspended in permeabilization buffer (0.1% saponin in D-PBS containing 5-mg/ml bovine serum albumin and 0.2% gelatin), immunolabeled with antibodies against NFATc2 (Thermo Scientific) or V5 tag (Invitrogen) and finally counter-stained with 4', 6-diamidino-2-phenylindole (DAPI) and anti-Alexa488 antibody (Life technologies).

The protein level of GAPDH, TCF-1, β-catenin, JunB and NFATc2 in ILC3s was detected by Western blot with the following antibodies: GAPDH (Beyotime, AF2819), TCF-1 (clone C63D9, Cell Signaling Technology, 2203), β-catenin (BD, 610154), JunB (clone C37F9, Thermo Fisher Scientific, PA1-835) and NFATc2 (clone 25A10.D6.D2, Thermo Fisher Scientific, MA1-025). In co-immunoprecipitation (co-IP) experiments, ILC3 pellets were resuspended in cell lysis buffer (Beyotime, P0013) including 1 mM PMSF (Beyotime, ST506). Then protein A + G magnetic beads (Beyotime, P2108), mouse IgG1 isotype control (clone G3A1, Cell Signaling Technology, 5415) and mouse anti-β-catenin (clone D10A8, Cell Signaling Technology, 8480) were used for IP. The IgG1-binding and β-catenin-binding proteins were further identified by Western blot.