Ecl chromogenic reagent

The total protein of each group was extracted using RIPA lysis buffer (Beijing solarbio science & technology co., Ltd, Beijing, China) according to the manufacturer’s instructions. Equal amounts of protein from each group of cells were subjected to SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked for 1 h at room temperature in 5% fat-free milk, the membrane was incubated with specific primary antibody at 4 °C overnight. The primary antibody was obtained from Abcam (Shanghai, China): Anti-LRP6 (Abeam, ab134146, 1:500). Then the membrane was incubated with an horse radishperoxidase (HRP)-labeled secondary antibody (Abcam, ab216773, 1:5000) at room temperature for 1 h. After the procedure of wash, the membrane was exposed with ECL chromogenic reagent (Millipore, Bedford, MA, USA), and then was exposed to film to observe the bands.

RIPA lysis buffer containing protease inhibitor Leupeptin (Roche, Basel, Swizerland) was used to prepare CG cell lysis solution. The protein sample obtained was separated by SDS‐PAGE and transferred onto nitrocellulose (NC) membrane. After being blocked with 5% skim milk, the membrane was incubated at 4℃ together with primary antibodies, including anti‐WIPF1 (1:1000, Abcam, ab132512), anti‐YAP (1:1000, Abcam, ab52771), anti‐TAZ (1:1000, Abcam, ab84927), and anti‐GAPDH (1:1000, Abcam, ab141703). The membrane was washed with TBST and then incubated together with horse radish peroxidase (HRP) conjugated secondary antibody (1:2000, Santa Cruz Biotechnology) for 1 h. After the membrane was exposed with ECL chromogenic reagent (Millipore, Bedford, MA, USA), automatic imaging system (ChemiDocXRS imaging system) was used for image acquisition, and the gray level was calculated.