Il 17rd

For flow cytometry experiments, cells were stained with anti-human IL-17RA (Biolegend), IL-17RB (R&D), IL-17RC (R&D), IL-17RD (R&D), IL-17RE (Novus) or the appropriate isotype control antibodies. Samples stained with unconjugated primary antibodies were then incubated with the appropriate conjugated secondary antibodies (Life technologies) before acquisition on an LSR II flow cytometer (BD). For Western blot analysis, vascular cell monolayers were washed in ice cold PBS twice and lysed in RIPA buffer (Sigma). Samples were then mixed with Laemmli's sample buffer and heated at 95°C for 10 minutes prior to loading. After electrophoresis, samples were transferred onto a PVDF membrane for 90 minutes at 4°C. Membranes were blocked with TBST containing either 5% BSA or 5% non-fat dry milk, and anti-human IL-17RA (Cell signaling), IL-17RC (Novus), or β-actin (Sigma) were added for overnight incubation at 4°C. Bound antibodies were visualized with anti-mouse or anti-rabbit HRP-conjugated antibodies (ThermoScientific) and SuperSignal Femto or Pico West (Pierce). Densitometry was performed using NIH Image J software.

Protein extraction and Western blotting were performed as previously described (24 (link)). Primary antibodies against IL17RD, FLAG, pAKT, pERK, pERBB2 EGFR, pEGFR, and beta actin were used with a dilution of 1:500 for IL17RD (R and D systems), 1:1000 for FLAG (Clontech, Mountain View, CA),1:500 pAKT (Cell Signaling Technology, Danvers, MA), 1:500 for EGFR (Cell Signaling Technology), 1:100 for pEGFR (Santa Cruz Biotechnology, Dallas, TX), 1:2500 for pERK (Santa Cruz Biotechnology), 1:200 for pERBB2 (Santa Cruz Biotechnology) and 1:5000 for beta actin (Sigma Aldrich, St. Louis, MO). Mean expression between treatment group was compared using a Student’s t-test.