Cw2334s

For the extraction of total protein, 100 μL RIPA lysis buffer (CW2334S; CWBIO, Beijing, China) and 1 μL Protease Inhibitor Cocktail (CW2200S; CWBIO, Beijing, China) were added to 100 mg of jejunal tissue, and the total protein was quantified using a bicinchoninic acid protein assay kit (CW0014; CWBIO, Beijing, China). Protein was separated by SDS-PAGE (Solarbio, Beijing, China) (20 μg per lane) and then transferred onto PVDF membranes (IPVH00010; Millipore, Danvers, MA). Next, the membranes were incubated with polyclonal goat anti-Wnt3 (ab116222; Abcam, UK), polyclonal rabbit anti-β-catenin (ab6302; Abcam, UK), monoclonal rabbit anti-TCF4/TCF7L2 (2565; CST, Boston, MA), polyclonal rabbit anti-ZO-1 (61-7300; Invitrogen, Camarillo, CA), and polyclonal rabbit anti-Claudin (ab129119; Abcam, UK) (all 1:2000 in Tris-buffered saline with Tween) overnight at 4°C. The membranes were incubated with HRP-conjugated rabbit anti-goat IgG (H&L) (1:5000; bs-0294R; Beijing Bioss Biotechnology Co., Ltd., Beijing, China) or HRP-conjugated goat anti-rabbit IgG (H&L) (1:5000; K008; Kmbio, Beijing, China) for 1 h at 37°C. Polyclonal anti-β-actin (1:5000; ab119716; Abcam, UK) was used for normalization of band intensities. The densitometric values of obtained immunoblot signals from 6 separate experiments were determined using Image J (National Institutes of Health, New York).