Ab216432

The protein for western blot analysis was extracted from skin tissues and concentration was determined using a BCA protein assay kit (Cat No: FD2001, Hangzhou Fude Biological Technology Company, China). The primary antibody included VEGFR (1:1000, ab39638, Abcam, UK), α-SMA (1:1000, ab124964, Abcam, UK), collagen I (1:1000, ab270993, Abcam, UK), collagen III (1:1000, ab7778, Abcam, UK), TGFβ1 (1:2000, ab215715, Abcam, UK), Smad3 (1:2000, ab40854, Abcam, UK), Smad2 (1:2000, ab40855, Abcam, UK), P-smad3 (phospho T179, 1:2000, ab74062, Abcam, UK), P-smad2 (phospho S467, 1:2000, ab280888, Abcam, UK), TIMP1 (1:2000, ab216432, Abcam, UK), GAPDH (1:4000, 2118, Cell Signaling Technology, Boston, USA) and Anti-rabbit secondary antibody (1:4000, ab6721, Abcam, UK). Proteins were visualized through a Clarity TM Western ECL Substrate (170–5061; Bio-Rad Laboratories Inc., USA) and a Tanon 5200 fully automatic chemiluminescence image analysis system (Tanon Science and Technology Co. Ltd., Shanghai, China).

Protein was isolated from tissues and cells using Cell lysis buffers (Takara, Dalian, China) following the suggestion of the manufacturer. Subsequently, the protein was separated by 10% SDS-PAGE gel, transferred with PVDF membranes, and blocked with 5% non-fat milk. After that, the membranes were incubated with rabbit anti-MMP9 antibody (1:1000; ab38898, Abcam, Cambridge, MA, USA), rabbit anti-TIMP1 antibody (1:1000; ab216432, Abcam, Cambridge, MA, USA), rabbit anti-CEACAM1 antibody (1:1000; #14,771, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-GAPDH antibody (1:2000; ab8245, Abcam, Cambridge, MA, USA) overnight at 4°C. The membranes were further incubated with HRP-labeled goat anti-rabbit IgG (1:2000; ab7090, Abcam, Cambridge, MA, USA) at room temperature for 1 h. Finally, bands were visualized using an enhanced chemiluminescence reaction solution (Bio-Rad, Hercules, CA, USA). GAPDH is used as an internal reference gene to normalize protein levels.

IL-1β was purchased from PeproTech Co., Ltd (Rocky, USA). The dulbecco's modified eagle medium-F12 (DMEM/F12) and phosphate buffer saline (PBS) were obtained from HyClone Co. (Logan, USA), while fetal bovine serum (FBS) was ordered from Gibco Co. (Detroit, USA). The TRIzol reagent was purchased from Invitrogen Co. (Carlsbad, USA). The SYBR Green dye and reverse transcription kits were purchased from Servicebio Co., Ltd (Wuhan, China). The antibodies for ribonucleotide reductase regulatory subunit M2 (RRM2, A5255), tissue inhibitor of metalloproteinases 1(TIMP1, A1389), and cyclin dependent kinase 1(CDK1, A0220) were ordered from ABclonal Technology Co., Ltd. (Wuhan, China). The aurora kinase B (AURKB, ab2254) and tissue inhibitor of metalloproteinases 1(TIMP1, ab216432) were purchased from Abcam plc. (Cambridge, UK). The SA-β-gal staining kits were obtained from Beyotime Co., Ltd. (Shanghai, China). The Annexin V-FITC/7-ADD apoptosis detection kits (Catalog Number: APK10448-F) were ordered from Sino Biological Co., Ltd. (Beijing, China). All primers were Tianyi Biotech Co., Ltd. (Wuhan, China).