Phospho tyr 689 stat2

Whole-cell extracts for immunoblotting were prepared by incubating cells for 10 min in RIPA lysis buffer (Thermo Fisher Scientific) with 50 mM dithiothreitol and Protease/Phophatase inhibitor cocktail (Cell Signaling Technology). For coimmunoprecipitation assay, cells were lysed in 50 mM Tris, pH 6.8, 0.5% NP-40, 200 mM NaCl, 10% glycerol, 1 mM EDTA, and 1× Protease/Phophatase inhibitor cocktail (Cell Signaling Technology). Cell lysates were incubated with V5 conjugated with protein G dynabeads for 2 h at room temperature. Immunoprecipitates were subjected to Western blotting. Immunoblotting was performed using the Bio-Rad Western blot workflow. Membranes were blocked in 5% BSA for primary antibodies or 5% nonfat dry milk for secondary antibodies. Antibodies used were STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Millipore), USP18 (Cell Signaling Technology), β-actin (ABclonal), and GAPDH (Millipore). Signal was detected with enhanced chemiluminescence detection reagent (SuperSignal West pico; Thermo Fisher Scientific) by film development.

For the experiment shown in Figure 2, cells were lysed in RIPA buffer (Thermo Fisher Scientific), DTT and a protease/phosphatase inhibitor cocktail (Cell Signaling Technology) and subjected to western blotting. For the experiment shown in Figure 5, cells were harvested in RA1 lysis buffer (Macherey-Nagel, Düren, Germany) containing 1% β-mercaptoethanol. The resulting cell lysates were stored at −20°C until RNA extraction and qPCR analysis. The antibodies used were directed against STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Millipore), USP18 (Cell Signaling Technology), ISG15 (Santa Cruz Biotechnology), β-actin (Cell Signaling Technology), and IFIT1 (Cell Signaling Technology). An enhanced chemiluminescence detection reagent was used for detection (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific).