Hil 20

Dulbecco’s modified Eagle medium (DMEM) and Dulbecco’s phosphate buffered saline (DPBS), fatal bovine serum (FBS), TrypLE^TM Express, pen-strep, glutamax, and protease inhibitor were purchased from Gibco Life Technologies (Grand Island, NY, USA). Human (h) IL-22, hIL-24, and all phosphorylated and secondary antibodies (Section 4.2) were procured from Cell Signaling Technology (Danvers, MA, USA). β-actin was purchased from Novus Biologicals (Littleton, CO, USA). The phosphatase inhibitor PhosSTOP was obtained from Roche (Basel, Switzerland). hIL-20 was purchased from R&D Systems (Minneapolis, MN, USA) and mIL-22 from PeproTech (Rocky Hill, NJ, USA). The specific inhibitor of STAT1 (Fludarabine) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), those of STAT3 and STAT5 from Calbiochem (San Diego, CA, USA), PI3K/Akt inhibitor (LY294002) and MEK1/2-ERK1/2 inhibitor (U0126) from Sigma-Aldrich (St. Louis, MO, USA), and p38 MAP kinase inhibitor (SB203580) from Tocris Bioscience (Bristol, UK).

PC–3 cells (2×10⁵) were stimulated with hIL–20 (200 ng/ml) (R&D Systems) for the indicated times. Total cell lysate was collect by adding 1× RIPA buffer containing phenylmethanesulfonyl fluoride (PSMF) (RIPA:PSMF = 10:1) and centrifuged at 13000 rpm at 4°C to collect the supernatant. Western blotting with antibody specific for phosphor-p38, phosphor-extracellular signal-regulated kinase (ERK1/2), phosphor-AKT, and phosphor-nuclear factor-kappa B (NF-κB) (Cell Signaling Technology) was used following the manufacturer’s instructions. β-actin was used as an internal control (Cell Signaling Technology). Quantification of proteins in western bands was done using ImageJ software.