Phospho stat1

The iPS-ML cells (1x10⁶/mL) were cultured with 50 ng/ml of IFNγ for the time exhibited. In some cells, 1 µM tofacitinib were added. Cells were lysed with dithiothreitol and incubated at 95°C for 5 min. Proteins were then separated by using SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. After blocking with Blocking buffer (nacalai tesque, Kyoto, Japan), immunoblotting was performed with monoclonal antibodies to phospho-STAT1 (Cell Signaling Technology, Danvers, MA) and β-actin (MBL, Tokyo, Japan) and exposed with ECL Western Blotting Reagent (Cytiva, Marlborough, MA).

PBS- and EV-treated K562 cells were lysed in RIPA buffer (Pierce) supplemented with a protease inhibitor cocktail (ThermoFisher Scientific). Cell and EV lysates were analyzed by SDS-PAGE and transferred onto PVDF (Bio-Rad). Membranes were blocked using 3% BSA and probed with specific antibodies. Proteins were detected using either Clarity (Bio-Rad) or WesternBright (Advansta) western ECL blotting substrates via chemiluminescence (ChemiDoc XRS+, Bio-Rad). Protein loading was normalized using anti-β-actin antibody (A5441, Sigma). Antibodies used for this study include: Alix (#2171), annexin V (#8555), Bcl-2 (#2872), CD9 (#13174), cleaved caspases -3 (#9664), -7 (#8438), -8 (#9496), and -9 (#9505), granzyme A (#4928), granzyme B (#4275), HSP70 (#4876), HSP90β (#5087), ICAM1 (#4915), LAMP-1 (#3243), phospho-STAT1: Ser727 (#9177) and Tyr701 (#9167), STAT1 (#9172), and VCAM1 (#13662) - (Cell Signaling); cytochrome c (sc-13156), full-length caspases -3 (sc-7272) and -7 (sc-28295), granulysin (sc-271119), MHC-1 (sc-55582), perforin-1 (sc-136994), Tsg101 (sc-136111), DNAM-1 (sc-376736) - (Santa Cruz); CD63 (SBI); rabbit polyclonal anti-LAMP-1 tail antibody; mouse monoclonal NKLAM and MHC-II antibodies made in-house. Horseradish peroxidase-labeled secondary anti-mouse and anti-rabbit antibodies were from Cell Signaling.

Cell lysates were prepared using M-PER Mammalian Protein Extraction Reagent with a cocktail of protease and phosphatase inhibitors (Cell Signaling). The protein samples were separated on an NU-PAGE Bolt Bis-Tris Plus Gels (Invitrogen), transferred onto nitrocellulose membranes (Bio-Rad), blocked using Intercept (TBS) Blocking Buffer (Li-Cor Biosciences), and then incubated overnight with different antibodies (Strange et al., 2019 (link)). Specific primary monoclonal antibodies used were rabbit anti-human against RIG-I (Cell Signaling Cat. No: 3743S), MDA5 (Invitrogen Cat. No: 700360), MXA (Santa Cruz Biotechnology Cat. No: sc-271024), IRF3 and phospho-IRF3 (Cell Signaling Cat. No: 11904S and Cat. No: 4947S), STAT1 (Cell Signaling Cat. No: 9172S), phospho-STAT1 (Cell Signaling Cat. No: 9167L), STAT2 (Cell Signaling Cat. No: 72604S), phospho-STAT2 (Cell Signaling Cat. No 4441) and mouse anti-human β-actin (Sigma Cat. No. A2228-200UL), all at 1:1,000 dilution. Secondary antibodies (1:10,000 dilution) were conjugated with IRDye 800 and IRDye 680 (Li-Cor Biosciences), and blots were scanned using an Odyssey infrared imager.

Liver tissues and cellular proteins were extracted with ice-cold lysis buffer [1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 137 mM sodium chloride, and 20 mM Tris (pH 7.4)]. Proteins (20 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride nitrocellulose membranes. Phospho-STAT1, STAT1, phospho-STAT6, STAT6, phospho-AMPK, AMPK, phospho-AKT, AKT, Bcl-2, Bcl-xl, and β-actin rabbit mAbs (Cell Signaling Technology, MA, USA) were used. HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology, MA, USA) was used as the secondary antibody.

Cells were homogenized in RIPA lysis buffer containing 1% protease inhibitor cocktail (Roche) to extract total protein. Equal amounts of protein homogenates were separated by SDS-PAGE (Bio-Rad) and transferred onto polyvinylidene difluoride membranes (pore size 0.45 μM, Bio-Rad), which were then blotted with monoclonal antibodies against GAPDH (1:1000, ZSGB-BIO), p38, phospho-p38, STAT1, and phospho-STAT1 (1:1000, Cell Signaling Technology). Proteins were detected with corresponding horseradish peroxidase-tagged secondary antibodies and enhanced chemiluminescence Western blot reagents (Advansta) and visualized using an enhanced chemiluminescence system (AlphaEase FluorChem System, Alpha Innotech Corp.).