Immunoprecipitation and immunoblot analysis.

Isolated IECs, tissues, or intestinal crypts or organoids were washed with ice-cold PBS and then homogenized on ice in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM sodium vanadate, 50 mM NaF, and a protease inhibitor cocktail (Nacalai Tesque). The lysates were centrifuged at 17,500 × g for 30 min at 4°C, and the resulting supernatants were subjected to immunoprecipitation and immunoblot analysis as previously described (22).