Incb018424

Protein levels of IL-27Rα in hPMSCs were determined using Western blot with CD3⁺T cells serving as a positive control. Expressions of IL-27Rα in hPMSCs as determined on different culture days for one generation were then analyzed by Western blot, as were levels of phosphorylated STAT1 (P-STAT1) and STAT1 in these hPMSCs. HPMSCs were pretreated with the Janus kinase 1/2 (JAK1/2) inhibitor INCB018424 (20 ng/ml, Selleck, Shanghai, China) for 1 h before stimulation with IL-27 and incubated in the presence or absence of INCB018424 for an additional 1 h; P-STAT1 and STAT1 expressions were then measured by means of Western blot. After adding RIPA lysis buffer to hPMSCs, the cells were lysed on ice for 40 min, centrifuged, subjected to SDS-PAGE electrophoresis, and then transferred to PVDF membranes. Rabbit anti-human IL-27Rα (Bioss, Beijing, China), β-actin (Bioworld, Nanjing, China), phosphorylated STAT1 (Abcam, Cambridge, UK), or STAT1 (Proteintech, Wuhan, China) antibodies were incubated with membranes at 4 °C overnight. Secondary goat anti-rabbit antibody (1: 1000) (Santa Cruz, CA, USA) was added on the following day. Blots were further washed and developed with enhanced chemiluminescent substrate (Beyotime, Shanghai, China), and protein bands were then visualized using a Western blot imager.

In this study, the following antibodies were mainly used: anti-flag (Protein Tech, Wuhan, 66008-3-IG), anti-STAT2 (Cell Signaling Technology, Danvers, MA, USA, 72604), anti-pSTAT2 (Cell Signaling Technology, Danvers, MA, USA, 88410), and anti-β-actin (TransGen Biotech, Beijing, HC201). The antibodies against NP protein of IAV and GE of PRV were produced by our laboratory. IFN-β was purchased from Sino Biologicals (Beijing), SB203580, INCB-018424, and CP690550 were from Selleck (Houston, TX, USA).

GSK2118436 (S2807), INCB018424 (S1378), LY2228820 (S1494), LY294002 (S1105), PLX-4032 (S1267), and SB202190 (S1077) were purchased from Selleckchem. GF-120918 (HY-50879) and KO-143 (HY-10010) were purchased from MedChemExpress. All compounds were used following 24 h serum withdrawal in low serum conditions (1% FBS) at the indicated concentrations and time.
Other drugs used were CHX (100 μg/ml) (Sigma–Aldrich), mithramycin A (200 nM) (Merck Millipore), λ−protein phosphatase (100U) (New England BioLabs), and MG-132 (100 nM) (Sigma–Aldrich).