Ab186421

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).

RIPA lysis buffer was used to extract proteins from tissues or cell lines and protein samples (50 μg) were resolved by SDS-PAGE and then probed with indicated antibodies. CYB561D2, FAS ligand (FASLG), TGF-β2 (TGFB2), CD70, PD-L1, PD-L2, CCL2, TDO2, total STAT3, p-STAT3 (Tyr705) antibodies were from Sigma-Aldrich (SAB2500281), abcam (ab15285), abcam (ab36495), abcam (ab77868), abcam (ab205921), abcam (ab187662), abcam (ab186421), abcam (ab123403), CST (4904) and CST (9131), respectively. STAT3 inhibitor C188-9 was from selleckchem (S8605) [16 (link)]. The band density was analyzed with Image J and the density of target protein is normalized by Tubulin band. There are four biological replicates for each WB experiment.