Immunoprecipitation ip lysis buffer

Cycloheximide (CHX; Sigma-Aldrich), anti-DYKDDDDK (Flag) G1 Affinity Resin (GenScript), phenylmethylsulfonyl fluoride (PMSF) (Gold Bio), immunoprecipitation (IP) lysis buffer (Thermo Scientific), TPCK-treated trypsin (Sigma-Aldrich), proteasome inhibitor MG132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, Selleck chem), NH₄Cl (Ammonium chloride, Selleck chem), and recombinant human IFN-α2 (GenScript) and IFN-γ (GenScript) were purchased from the indicated manufacturers. Antibodies against JAK1, STAT1, phospho-STAT1, and β-actin were purchased from Sigma-Aldrich; antibodies against SOCS1 and SOCS3 were purchased from GeneTex, antibodies against influenza virus NP, M1, and NS1 were purchased from GeneTex; antibodies against DYKDDDDK (Flag) tag and HA tag were purchased from Cell Signaling Technology. Human SOCS1 siRNAs si-1, GCAUCCGCGUGCACUUUCAdTdT, and si-2, CUACCUGAGCUCCUUCCCCdTdT were synthesized by Gene Pharma.

Cells were harvested and washed twice with phosphate-buffered saline (PBS), lysed with immunoprecipitation (IP) lysis buffer (Thermo Scientific, MA, USA) for 30 min on ice, and centrifuged for 20 min at 12,000 × g at 4 °C. Whole-cell lysates were subjected to SDS–PAGE and transferred onto nylon membranes. Membranes were blocked with 5% nonfat milk (or 5% BSA) for 1 h at room temperature, and then incubated with specific primary antibodies at 4 °C overnight. They were then washed with PBS-T three times and subsequently hybridized with peroxidase-conjugated secondary antibodies for 1 h at room temperature, followed by washing with PBS-T. Finally, the ChemiDoc XRS system (Bio-Rad, USA) and Image Lab Software were used for visualization of blots.