Ab68185

The tissues were fully lysed and sonicated, and the total protein was extracted. Protein levels were quantified using a BCA Protein Assay Kit. Polyacrylamide gel electrophoresis was performed with 10 μg protein from each sample. Next, proteins on the gel were transferred onto a polyvinylidene fluoride (PVDF) membrane by semi-dry transfer, and the PVDF membrane was sealed with 5% skim milk powder for 2 h at RT. Thereafter, the membrane was probed with the primary antibody at 1:1000 dilution with phosphate buffer saline (PBS) against matrix metalloproteinase 8 (MMP8), MMP9, IL-17β, tissue inhibitor of metalloproteinase 1 (TIMP1), Ccl2, Ccl7, TNF ligand superfamily member 9 (TNFSF9), and Fas apoptotic inhibitory molecule (FAIM), S100a8, S100a9 at 4 °C overnight. All primary antibodies were provided by Cell Signaling. The PVDF membrane was then washed three times with PBS-Tween for 8 min each. The secondary antibody was diluted with PBS and was incubated for 1 h at RT, and the PVDF membrane was washed again. β-actin was used as a loading control. Proteins were visualized using an enhanced chemiluminescence solution. The following antibodies were used: MMP8 (Abcam, ab81286), MMP9 (CST, 13667T), IL-17β (Abcam, ab125029), TIMP1 (CST, 8946S), Ccl2 (Abcam, ab186421), Ccl7 (Abcam, ab182793), TNFSF9 (Abcam, ab68185), FAIM (Abcam, ab154570), S100a8 (CST, 47310T), and S100a9 (CST, 73425S).