Anti il 17ra

Anti-CD31 (390), mAbs from ThermoFisher; PB- & BV711-anti-mouse CD45 (30-F11 dilution 1/500), AF488-anti-CD115 (AFS98 dilution 1/300), PE-Cy7-anti-CD31 (390 dilution 1/300), AF488- & AF647-anti-CD54 (YN1/1.7.4 dilution 1/300), APC-anti-CD140b (18A2 dilution 1/300), APC-Cy7-anti-CD115 (AFS98 dilution 1/300), AF647- & PE-Cy7-anti-CD117 (2B8 dilution 1/300), PB-FcεRI (MAR1 dilution 1/300), AF647-anti-IL-17A (TC11-18H10.1 dilution 1/300), AF594-anti-CD4 (GK1.5 dilution 1/300), AF700-anti-CD3 (17A2 dilution 1/300), BV605-anti-CD41 (MWReg30 dilution 1/300), PE-anti-CD49d (R1-2 dilution 1/300), AF647-anti-CD11c (N418 dilution 1/300), Rat IgG1 mAbs from Biolegend; Anti-IL-17RA (G-9 dilution 1/100) from SantaCruz; Desmin (D33 dilution 1/100) were obtained from Dako; Anti- αSMA (1A4 dilution 1/300) from Sigma-Aldrich; anti-mouse CXCL1 (polyclonal dilution 1/100) from R&D systems. Anti-mMCP-1 (RF6.1 dilution 1/100) from ThermoFisher. Anti-MRP14 mAb (dilution 1/500) was a gift form Dr Nancy Hogg (The Francis Crick Institute, UK).

Cells were lysed by radioimmunoprecipitation assay (RIPA) buffer (50 mM sodium fluoride, 0.5% NP-40, 10 mM sodium phosphate monobasic, 150 mM sodium chloride, 25 mM Tris pH8.0, 2 mM ethylenediaminetetraacetic, and 0.2 mM sodium vanadate) with a fresh supplement of 1× protease inhibitor cocktail (cat# P8849, Millipore Sigma, St. Louis, MO, USA). Around 80 µg of whole cell lysate was loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to the polyvinylidene fluoride membranes (PVDF, cat# 83-646R, Genesee Scientific, San Diego, CA, USA). Incubation of primary antibodies (anti-GAPDH (cat# AB2302, 1:5000 dilution, Millipore Sigma, St. Louis, MO, USA), anti-IL17RA (cat# sc-376374, 1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PLK4 (cat# 12952-1-AP, 1:1000 dilution, Proteintech, Rosemont, IL, USA)) was conducted at room temperature for 1.5 h. The IRDye^® 680RD Goat anti-Mouse IgG Secondary Antibody (Li-COR, Lincoln, NE, U.S.A) was incubated at room temperature for 45 min with a dilution ratio of 1:10,000. Blots were scanned with an Li-COR Odyssey 9120 Digital Imaging system.