Gapdh

Immunofluorescence staining was used to assess the expression of Col-II and aggrecan (Zen-bio, Sichuan, China) in the NPCs. Western blotting was used to evaluate the expression of Col-II, aggrecan, MMP-3 (Proteintech, Wuhan), MMP-13 (Proteintech, Wuhan), and GAPDH (Immunoway, Jiangsu). Subsequently, RT-qPCR with fluorescence detection was performed to assess the expression of ECM-related genes. Expression was analyzed using the 2^−ΔΔCt method.

Total protein from skin tissues was extracted using RIPA buffer containing phosphatase and protease inhibitors. Lysates were then centrifuged at 12,000 g for 10 min. Protein concentrations were measured using the Pierce BCA Protein Determination Kit (Cat. No.23227, Thermo Scientific, USA). 50 μg of total protein/well were then separated using a 10%/12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. PVDF membranes were blocked in 5% skim milk for 1 h at room temperature and then incubated with primary antibodies against GAPDH (1:5,000, ImmunoWay Biotechnology Company, USA), SP (1:1000, Cell Signaling Technology, USA); or NK1R (1:2000, Cell Signaling Technology, USA) overnight at 4 °C. Membranes were then washed with Tween 20 wash buffer and then incubated with secondary anti-rabbit IgG (H + L) (111-035-003, Jackson Immuno Research Laboratories, West Grove, PA, USA; 1:10,000) or goat anti-mouse IgG (H + L) (115-035-003, Jackson ImmunoResearch Laboratories; 1:10,000) at room temperature for 1 h. Immunofluorescence was measured using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Specific protein bands were quantified using the ImageJ one analysis software.

Cells were lysed in ice-cold RIPA buffer, and 20 µg protein was separated by electrophoresis on 8–12% denaturing SDS-PAGE gels. The blots were probed with primary antibodies overnight at 4ºC, followed by incubation with the appropriate secondary antibodies at room temperature for 1 h. The antibodies used in this study included DLX1 (Cat: 13046-1-AP, Proteintech) and GAPDH (Cat: YM3029, Immunoway).

Immediately after the animals were sacrificed, the flap tissue of area II was collected and analyzed by Western blot. For Western blot analysis, each sample was treated with lysis buffer to release tissue proteins and the protein concentration was measured by a BCA method. Next, gel electrophoresis was used to separate the proteins, which were then electrotransferred to PVDF membranes and blocked with nonfat milk 5%. The samples were eluted with TBST three times and incubated at 4 °C after adding the primary antibody solution. The incubation concentration of the primary antibody is as follows: Bax (1:1000, Immunoway), Bcl-2 (1:1000, Immunoway), β-actin (1:5000, Immunoway), VEGF (1:1000, Immunoway), Nrf2 (1:1000, Immunoway), LC3B(1:2000, Abcam), SQSTM1/p62 (1:30,000, Abcam), CTSD (1:5000, Abcam), GAPDH (1:5000, Immunoway). Subsequently, secondary antibodies conjugated to HRP were incubated with the samples for 2 h at room temperature. Finally, an ECL kit was used to visualize the bands on the membrane, and the intensity of the bands was quantified using Image Lab software.