Alpha imager scanner

For western blotting, proteins from each sample were separated by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Cell Signaling Technology, MA, USA) at 300 mA for 100–120 min. The membrane was initially blocked in 5% bovine serum albumin for 2 h at room temperature and then incubated overnight at 4°C with primary heat shock protein- (HSP-) 70 (1 : 2000, Abcam, UK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1 : 1000, Cell Signaling Technology, MA, USA), histone deacetylase 5 (HDAC5) (1 : 1000, CST, USA), or integrin alpha-5 (ITGA5) antibody (1 : 1000, Cell Signaling Technology, MA, USA), respectively, followed by three or four washes with Tris-buffered saline, 0.1% Tween 20. The membrane was then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1 : 3000, Beyotime, China) for 2 h at room temperature. Protein expression was then observed by chemiluminescence using an Alpha Imager scanner (Tecan, Thermo Fisher Scientific, USA).

Skin wound tissues from each group on day 14 and day 21 were isolated, and samples were washed with PBS. Then, the tissue lysate was added to completely lyse the tissue, and the lysate was centrifuged at 1,200 g for 10 minutes to collect the supernatant solution containing the protein. Supernatants containing equal amounts of protein were added to a 10% SDS-PAGE gel, were electrophoresed, and were transferred to a PDVF membrane. The membrane was then blocked in 5% BSA for 2 h at room temperature and incubated overnight at 4°C in the primary antibody solution. The primary antibodies recognized GAPDH (Cell Signaling Technology, MA, USA) and EGFR (Cell Signaling Technology, MA, USA). Finally, the membrane was washed 3-4 times with TBS-T and incubated with a goat anti-rabbit HRP-conjugated secondary antibody (Cell Signaling Technology, MA, USA) for 2 h at room temperature. The intensity of protein expression was observed on an Alpha Imager scanner using chemiluminescence (Tecan, Thermo Scientific, USA), and expression analysis was performed using ImageJ software.

The cells from each sample were collected and lysed in radioimmunoprecipitation (RIPA) buffer (Thermo Scientific), followed by centrifugation at 1,200×g for 10 min and subsequent collection of the supernatant. Western blotting was performed as previously described (Xiong et al., 2019 (link)). The primary antibodies were GAPDH (1:20,000, Proteintech, China), Lgr5 (1:1,000, Abcam, United States), TGF-β1 (1:1,000, Santa Cruz Biotechnology, United States), and β-catenin (1:1,000, Cell Signaling Technology, United States), and the secondary antibody was horseradish peroxidase-conjugated goat antimouse (1:10,000, Abcam, United States). Protein expression was observed and visualized by chemiluminescence using an Alpha Imager scanner (Tecan, Thermo Scientific).