Sc 166699

HEK293T cells, L cells (an immortalized mouse fibroblast cell line), SW480 cells and HCT116 cells were obtained from Shanghai Life Academy of Sciences cell library (Shanghai, China). All cells were grown in DMEM medium (Invitrogen, Carlsbad, CA), and maintained in culture supplemented with 10% heat-inactivated fetal calf serum, 100 ug ml⁻¹ of penicillin, and 100 μg ml⁻¹ of streptomycin at 37 °C with 5% CO₂ in a humidified incubator (Thermo, Waltham, MA). During the study, all cell cultures were periodically tested for mycoplasma by using MycoAlert Mycoplasma Detection Kit (Lonza, Rockland, ME). For western blot, antibodies specific for VGLL4 (1:500, ab140290), TEAD4 (1:500, ab58310), Histone H3 (1:1,000, ab6002) and β-catenin (1:1,000, ab2365) were purchased from Abcam (Cambridge, UK); those for FLAG (1:5,000, M4439) and α-tubulin (1:2,000, T6199) were from Sigma (St. Louis, MO); and those for TCF4 (1:500, sc-166699), c-Jun (1:1,000, sc-4113) and GST (1:1,000, sc-138) were bought from Santa Cruz Biotechnology (Santa Cruz, CA).

Pancreatic tissues were fixed for 4 h in 4% paraformaldehyde in PBS and embedded for paraffin sectioning (5 μm). The sections were deparaffinised, rehydrated, and incubated overnight at 4°C with goat antisera against insulin, TCF7L2 antibody (1:60–70, D-4, sc-166699, Santa Cruz, CA, United States), and DAPI (AR1176, Wuhan Boster Company). The sections were subsequently probed with secondary antibodies for 20 min at 37°C (Yang et al., 2012 (link)). Images of the pancreatic tissues were acquired using a fluorescent inverted microscope (Olympus IX71, Japan). For morphometric analysis, the fluorescence intensity of pancreatic sections was quantified using the Image J 1.37c¹.

Total protein was extracted from the renal cortex of rats and quantified with the BCA assay (MultiSciences Biotech Co., Ltd., Hangzhou, China). Then the protein was diluted with 5× loading buffer and denatured at 100°C for 5 minutes. Afterwards, the protein samples were electrophorised in SDS polyacrylamide gel and then transferred onto PVDF membranes (Millipore Corporation, Bedford, MA, USA). The nonspecific background bindings were blocked with 5% nonfat milk for an hour. Then the membranes were incubated in primary antibodies at 4°C overnight and secondary antibodies for 1 hour. Optical density of the bands was scanned by Odyssey infrared fluorescence imaging system (LI-COR, USA) and quantified using ImageJ (National Institutes of Health, USA). The primary antibodies used are as follows: Wnt4 (sc-376279, Santa Cruz Biotechnology, Inc., USA, 1: 500), TCF4 (sc-166699, Santa Cruz Biotechnology, Inc., USA, 1: 500), GSK3β (ab93926, Abcam, Inc., UK, 1: 1000), p-GSK3β (ab131097, Abcam, Inc., UK, 1: 1000), E-Cadherin (GTX100443, GeneTex Inc., CA, USA, 1: 1000), ɑ-SMA (ET1607-43, Hangzhou HuaAn Biotechnology Co., Ltd., Hangzhou, China, 1: 5000), and Vimentin (ab92547, Abcam, Inc., UK, 1: 5000).