Odyssey imaging software 3

At the end of exposure, the explants were stored in −80°C with RNA later (Ambion, Grand Island, NY, USA). Tissues were homogenized, lysed, and the lysate (10 μg) was loaded on 10% acrylamide SDS-PAGE NEXT GEL (Amresco, Solon, Ohio), followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, Calif). The blots were then blocked for 1 h at room temperature and incubated overnight at 4°C with antibodies specific for IL-17A (R&D Systems, Minneapolis, MN, USA), IL-17 F (Santa Cruz Biotechnology, Santa Cruz, Calif) and GAPDH (Millipore, Temecula, USA). After washing (0.1% Tween-20/PBS), the membranes were incubated with a 1:15,000 dilution of IRDye 800 donkey anti-goat IgG and IRDye 680 goat anti-mouse IgG (Rockland) in blocking buffer and analyzed with an Odyssey IR scanner using Odyssey imaging software 3.0 (LI-COR Biosciences, Inc).

2×10⁶ B cells were starved using medium with 0.1% FBS for 18 hours. Cells were stimulated with 50 ng/ml IL-17A and IL-17F for 0, 10, and 20 minutes and total proteins were extracted using lysis buffer (1% Triton X-100 containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Protein lysates (10 µg) were then resolved on 10% acrylamide SDS-PAGE gel and blots were probed with antibodies to phosphorylated p38 MAPK (Millipore) and total p38 MAPK (Millipore). Membranes were analyzed with an Odyssey IR scanner using Odyssey imaging software 3.0 (LI-COR Biosciences, Inc).