High sensitivity chemiluminescence imaging system

The cell lysates were separated by 10–15% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was incubated with primary antibodies, including rabbit anti-ZBTB48 (Abcam, ab50588, Cambridge, United Kingdom), rabbit anti-GAPDH (Proteintech Co., 10494-1-AP, Wuhan, China), rabbit anti-P21 (Proteintech, 10355-1-AP), rabbit anti-P53 (Proteintech, 10442-1-AP), rabbit anti-CDKN2A/P16-INK4a (Bioss Co., bs-4592R, Beijing, China), rabbit anti-P14ARF (Bioss, bs0534R), rabbit anti-MDM2 (Bioss, bs-23748R), and rabbit anti-PPAR gamma (Bioss, bs-4590R) antibodies overnight at 4 °C, followed by incubation with an HRP-labeled goat or mouse anti-rabbit IgG for 1 h at room temperature. Specific proteins were detected by a high-sensitivity chemiluminescence imaging system (BioRad Laboratories, Hercules, CA, United States). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control.

After treatment, HaCaT cells and skin tissue were extracted using RIPA lysis solution (Cat. No. P00138; Beyotime) containing 1% PMSF to extract proteins and quantified using BCA protein kits (Cat. No. PC0020; Solarbio). Protein samples (35 μg) were separated by SDS-PAGE using A 12% resolving gel and then transferred to PVDF membranes (Millipore, United States). The PVDF membranes were blocked for 1 h in PBS containing 1% BSA, and the PVDF membrane was incubated overnight at 4°C with primary antibodies, including MMP-1, MMP-9, Bax (1:1,000; Cat. No. 2772; CST), Bcl-2 (1:1,500; Cat. No. ab196495; Abcam), cleaved caspase-3 (1:1,000; Cat. No. 9661; CST), and β-actin (1:5,000; Cat. No. AF5003; Beyotime). The membranes were then incubated with the appropriate goat anti-rabbit secondary antibodies for 1 h at RT, followed by three washes in TBST. Proteins were detected using the ECL reagent. The loading protein was normalized to β-actin, and the target protein were exposed using a high-sensitivity chemiluminescence imaging system (Bio-Rad, United States).