Adam17

Cells were harvested with trypsin-EDTA (Lonza) and washed in FACS buffer (PBS containing 1% FBS). 5×10⁵ cells were stained in a volume of 25μl for 30 minutes at 4°C. Antibodies were diluted in FACS buffer using the following concentrations: FITC conjugated a-HER2 affibody (1:1500, Bromma, Sweden); a-EGFR (1:2000, clone H11, DAKO, Carpinteria, CA); a-HER3 (1:1500, clone SGP1, Abcam); a-HER4 (1:200, clone H4.77.16, Abcam); a-IGFR (1:50, clone 33255, R&D, Minneapolis, MN); ADAM10 (1:500, MAB1427, R&D); ADAM17 (1:100, MAB9301, R&D). Secondary APC labeled a-mouse (550826, BD) was diluted 1:800. After washing, cells were resuspended in FACS buffer containing 50ng/ml propidium Iodide (PI) (Sigma) and acquired on a FACSCanto II (BD, Franklin Lakes, NJ). Data were analyzed with FlowJo 10 (Tree Star, Ashland, OR). The geometric mean fluorescence (gMFI) intensity in the relevant channel was calculated from the PI negative gate; gMFI from the isotype control was subtracted from the sample yielding the delta gMFI.

TNF (Cat. No. 430201, BioLegend, San Diego, CA, USA), TNFR1, TNFR2, TGβ-1, and TIM3 (Cat. No. DY225, DY726, DY240, and DY2365 respectively; R&D Systems, Minneapolis, MN 55413, USA), and ADAM17 (Cat. No. SEB555Hu 96T, Cloud-Clone Corp., Katy, TX, USA) were evaluated. All the molecules were quantified by an ELISA assay in the sera, complying with the manufacturer’s instructions.

Total proteins were extracted from PBMCs using RIPA lysis buffer (Solarbio, China), and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA). An equal amount of protein was loaded onto 10% SDS-PAGE and subjected to electrophoresis, then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was washed with Tris-buffered saline-Tween (TBST) (Solarbio, China), and blocked with 5% skim milk powder or bovine serum albumin (BSA) (Sigma-Aldrich, Germany). Incubation was carried out using specific primary antibodies against STAT3 (Abcam, U.K), pSTAT3 Y705 (Abcam, U.K), ADAM17 (R&D Systems, USA), IL-23 R (Abcam, U.K), and GAPDH (Santa Cruz, USA), followed by the incubation with the secondary antibodies HRP Goat anti-Mouse antibody (abclonal, USA) or HRP Goat Anti-Rabbit IgG (abclonal, USA). Pierce™ ECL Western Blotting Substrate (Thermo Fisher, USA) was used as the chemiluminescent substrate, and X-ray imaging was used to image the bands. Relative expression levels of proteins were quantified by densitometric values of fluorogram bands which was analyzed by Image J software (NIH, USA) and each value was normalized to those corresponding control protein band.

MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, and ADAM17 were purchased from R&D Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, and 930-ADB, respectively). All common chemicals were purchased from Sigma. Marimastat was purchased from Tocris (cat# 2631), actinonin was from Sigma-Aldrich (cat# 01809). Hybricare medium was from ATCC (ATCC® 46-X™).

MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-13, MMP-14, ADAM10, ADAM17 and Mca-KPLGL-Dpa-AR-NH2 fluorogenic peptide substrates were purchased from R&D Systems (cat # 901-MP, 902-MP, 908-MP, 911-MP, 910-MP, 511-MM, 918-MP, 936-AD, 930-ADB, and ES010, respectively). All common chemicals were purchased from Sigma. NFF449 was purchased from Tocris (cat# 1391) and actinonin was from Sigma-Aldrich (cat# 01809).