Anti tcf3

Corresponding sections of wound tissues from different groups were incubated with the following primary antibodies: anti-Tcf3 (diluted 1:200), (Abcam, MA, U.S.A.). IgG conjugated with peroxidase was used as the secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). In negative controls, incubation with the above antibodies was omitted. The sections were examined using an upright light microscope (Olympus, Tokyo, Japan). The immunohistochemical staining intensity and area were evaluated by three independent investigators.

Total proteins from cells or tissue samples were extracted using RIPA buffer containing protease inhibitor (Beyotime Institute of Biotechnology, China) on ice. For nuclear protein extraction, a CelLytic™ NuCLEAR™ extraction kit (Sigma, Darmstadt, U.S.A.) was used, according to the manufacturer’s instructions. The concentration of proteins in the sample was detected by a BCA protein assay kit (Thermo Fisher Scientific, MA, U.S.A.). The proteins were separated by 10% SDS/PAGE and electrotransferred to a PVDF membrane (Merck Millipore, Darmstadt, U.S.A.). The membrane was then blocked in 5% non-fat milk for 1 h and incubated overnight with primary antibodies at 4°C. The next day, the PVDF membrane was washed with 1× TBST, followed by incubation for 1 h with horseradish peroxidase (HRP)-labeled secondary antibodies diluted with 5% non-fat milk. Finally, the protein bands were detected using an ECL system (GE, U.S.A.). GAPDH and Histone were used as an endogenous control for total and nuclear proteins, respectively. The primary antibodies in the present study included the following: anti-GAPDH (1:5000, Sigma–Aldrich, Darmstadt, U.S.A.), anti-Histone H (1:5000, Abcam), anti-β-catenin (1:1000, Abcam), anti-CK-18 (Cytokeratin-18) (1:1000, Abcam), anti-CK-19 (1:1000, Abcam), anti-P63 (1:2000, Abcam) and anti-Tcf3 (1:2000, Abcam).

GFP-Id2^WT and GFP-Id2^ΔHLH Jurkat cells were generated by lentiviral infection. HEK-293 cells were transfected with the appropriate plasmids. For Co-IP, a Pierce coimmunoprecipitation kit was used. Briefly, cells were harvested and lysed with IP lysis buffer containing 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40 and 5% glycerol (Thermo Fisher). Cell lysates were then incubated with the indicated antibody (anti-GFP, Santa Cruz; anti-Flag, CST Signaling; anti-Tcf3, Abcam)-conjugated resin in spin columns (Thermo Fisher) overnight at 4 °C with rotation. The resin was then washed with IP lysis buffer two times, and proteins were eluted with elution buffer. The eluted samples were then used for immunoblotting with appropriate antibodies. The immunoblot images were obtained in a Bio-Rad ChemiDoc Imaging System.