Stat1

Western blot assay was carried out as follow: cells were lysed with RIPA buffer (Santa Cruz, USA) and cleared lysate was collected by centrifugation for protein separation on 10% SDS-polyacrylamide gel. The proteins were transferred onto PVDF membranes (Millipore) and detected with respective antibodies at 4°C overnight, followed by incubation with either IRDye Fluor 680-labeled IgG or IRDye Fluor 800-labeled IgG secondary antibody (Li-Cor Bioscience). The images were scanned and quantified by densitometric analysis by Li-COR Odyssey Infrared Imager. Primary antibodies against VP1 (Millipore), CD81 (Cell Signaling Technology), IRAK1 (Santa Cruz), CD63 (Abcam), ISG15 (Abcam) and Rab27a, TRAF6, STAT1, TSG101, BST-2/Tetherin, and GAPDH (all from Proteintech), VP2, 3AB, 3C and 3D (all from Genetex) were used.

Western blotting was performed as previously described [17 , 20 (link)]. Briefly, cells were collected, lysed, and then quantified with a BCA kit (Beyotime, China). 20 μg of protein was loaded into a SDS gel, followed by electronic transfer onto PVDF membranes (Millipore, USA), and then incubated with primary antibodies against DHX9 (No.17721–1-AP, Proteintech), STAT1 (No.10144–2-AP, Proteintech), STAT1 phospho Ser727 (p-STAT1, No.39634, Active motif), p38 (No.14064–1-AP, Proteintech), Phospho-P38 MAPK (Thr180/Tyr182) (p-p38, No.28796–1-AP, Proteintech), JNK (No.66210–1-Ig, Proteintech), Phospho-JNK (Tyr185) (p-JNK, No.80024–1-RR, Proteintech), ERK1/2 (No.11257–1-AP, Proteintech), Phospho-ERK1/2 (Thr202/Tyr204) (p-ERK1/2, No.80031–1-RR, Proteintech), p65 (No.66535–1-Ig, Proteintech), p65 (phospho S536) (p-p65, sc-136548, Santa Cruz), GAPDH (ab8245, Abcam), flag (ab205606, Proteintech), or Lamin B1 (No.66095–1-Ig, Proteintech). After incubating with the secondary antibodies, the protein bands were detected using an ECL kit (Pierce, USA). The experiment was repeated three times independently.

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.

CD3^-CD56⁺ dNK cells from the three groups were incubated for 36 h before harvesting. Equal amounts of protein from total-cell lysates were separated by 12% SDS-PAGE (Beyotime) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were then blocked at room temperature for 2.5 h in 7% nonfat dry milk in TBS-T buffer. Membranes were incubated with gentle rocking 1.5 h at room temperature with primary antibodies for Tim-3 (1/2000, Proteintech), GranzymeB (1/2000, Abcam), Perforin (1/1000, Proteintech), GranzymeA (1/600, Proteintech), PI3K (1/500, Proteintech), AKT (1/500, SAB), pAKT (1/500, SAB), STAT1 (1/500, Proteintech), STAT3 (1/600, Proteintech), pSTAT1 (1/500, Abcam), pSTAT3 (1/600, Abcam), IL-10 (1/500, Abcam), IFN-γ (1/500, Proteintech) and GAPDH (1/40000, Proteintech) as a loading control. Membranes were washed with TBS-T 5 times for 10 min each, then incubated with the appropriate secondary antibody for 2 h at room temperature. Then immune complex was visualized with an enhanced chemiluminescence (ECL) detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were determined by Image J software (Rawak Software, Inc., Germany).

Antibodies used in this study included p-ERK (Cat. #4370T, Cell Signaling Technology), ERK (Cat. #4695T, Cell Signaling Technology), p-STAT1 (Cat. #7649S, Cell Signaling Technology), STAT1 (Cat. #10144-2-AP, Proteintech), p-PKR (Cat. #ab32036, Abcam), Anti-phospho-PKR (Thr451) (07-886, Merck), PKR (Cat. #ab32506, Abcam), and anti-GAPDH (Cat. #2118S, Cell Signaling Technology).