Isg15

Cells plated at 2 × 10⁵cells/well/2 mL media were plated in a 6-well plate the night before IFN-α treatment as described. After 30 min, media were removed, and cells were lysed and collected using 150 μL 1X NuPAGE LDS Sample Buffer (Invitrogen, Thermo Fisher). Lysates were stored at −80°C. Lysate samples were directly sonicated with a microtip at 20% amplitude 3X for 5 s before being incubated at 95°C for 5 min. Lysate samples (10 μL) were electrophoresed on a Nu-PAGE 4%–12% Bis-Tris gel with MOPS buffer at 150 V for 70 min and transferred to a membrane using an iBlot2 gel transfer system. The membrane was blocked with 5% milk in PBS-T, incubated with primary antibodies overnight at 1:1,000 dilution at 4°C, followed by secondary antibody incubations at 1:10,000 dilution for 60 min at 25°C. Antibody binding was detected using Immobilon Western Chemiluminescent HRP Substrate and imaged using Protein Simple Fluor Chem R. The following antibodies were used for western blot analysis: Stat1 (#14994), Phospho-Stat1 Tyr701 (#9167), ISG15 (#2758) from Cell Signaling Technologies (Danvers, MA, USA), and beta actin HRP (#ab20272) from Abcam (Cambridge, MA, USA).