Radioimmunoprecipitation assay lysis buffer

After homogenization, all skin tissues were lysed for 30 min with ice-cold radioimmunoprecipitation assay lysis buffer (CoWin Biosciences, Taizhou, China). Protein lysate (15 μg; concentration determined by a BCA assay) was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; CoWin Biosciences, Taizhou, China) on a 6% gel for collagen I and collagen III; 10% gel for Aurora B, CK14, and CK15; 12% gel for PCNA, pH3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) at 80 V for 1.5 h, and then transferred to polyvinylidene fluoride membranes (Merck, Darmstadt, Germany). The membranes were blocked with 5% bovine serum albumin in PBS at room temperature for 1.5 h, followed by incubation with primary antibodies against collagen I (1:1000; Cell Signaling Technology, Danvers, MA, United States), collagen III (1:1000; Cell Signaling Technology, Danvers, MA, United States), PCNA (1:1000; Abcam), Aurora B (1:1000; Abcam), pH3 (1:200; Abcam), CK14 (1:1000; Proteintech), CK15 (1:2000; Proteintech), and GAPDH (1:20,000; Proteintech) at 4°C overnight. Quantitative analysis was performed on the immunoreactive bands using ImageJ software (v.1.53k; NIH, Bethesda, MD, United States).