C jun h79

Immunoblot analysis was performed after 8 or 12% SDS-PAGE, with overnight incubation with a 1:1,000 dilution of primary antibody and followed by a 1:5,000 dilution of horseradish peroxidase–conjugated anti–rabbit, anti–mouse, or anti–goat antibody (GeneTex). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). The homemade antibody against IL-17RB (A68) was used. Antibodies against CCL20 (67310), CXCL1 (20326), and IL-8 (6217) were purchased from R&D Systems. Antibodies against TFF1 (EPR3972), TAK1 (GTX107452), p84 (GTX118740), ATF2-phosphoThr71 (E268), ATF2 (E242), IKKα (GTX27609), IKKβ (GTX105690) and p65 (GTX102090) were purchased from Genetex. Antibodies against p-ERK1/2 (4370), ERK1/2 (4695), p-IKK (2687), c-JUN-phosphoSer63 (9261), Akt (9272), and p-Akt (4058) were purchased from Cell Signaling Technology. Antibodies against TRAF6 (Sc-8409), ACT1 (H300), AML1α (N20), and c-JUN (H79) were purchased from Santa Cruz Biotechnology. Protein concentration was determined by the Bradford assay (Bio-Rad Laboratories) before loading and verified by α-tubulin or GAPDH level using a 1:100,000 dilution of anti-tubulin antibody (GeneTex). The optical density was determined using the National Institutes of Health ImageJ program.

Total protein was isolated using the total protein extraction kit (BestBio Science), and the concentrations were measured using BCA Protein Assay Kit (Beyotime Biotechnology). Proteins were separated on the 10% SDS PAGE gels, and subsequently transferred to polyvinylidene fluoride membranes (PALL corporation). The membranes were probed with primary antibodies Fra-1 (R-20) (1:500 dilution) (Santa Cruz Biotechnology), c-Jun (H-79) (1:500 dilution) (Santa Cruz Biotechnology), or OLFML2A (1:500 dilution) (Abcam plc., UK) made from rabbit, and then probed with donkey anti-rabbit IgG antibody (1:10,000 dilution) (GE Healthcare Company). The protein levels were normalized by probing β -actin antibody (1:10,000 dilution) (Sigma-Aldrich LLC.) and sheep anti-mouse IgG antibody (1:10,000 dilution) (GE Healthcare Company). Immunosignals were imaged using a Tanon 5200 Multi Automatic Chemiluminescence / Fluorescence Image Analysis System (China).