Western lighting plus ecl system

Cells were lysed in RIPA buffer, and whole-cell extracts were quantified by the Bradford assay (Bio-Rad). For assessment of nuclear proteins, nuclear extracts were obtained using NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific). The protein samples or cell lysates were analyzed by SDS-PAGE and Western blot. Briefly, after proteins were transferred onto PVDF membranes (Millipore), the membranes were incubated with indicated primary antibodies, followed by a HRP-conjugated secondary antibody. Immunoreactive bands were detected using the Western Lighting Plus-ECL system (PerkinElmer) or the SuperSignal West Dura Extended Duration Substrate (Pierce). The primary antibodies used for Western blot included anti-p38 (2F11, Millipore), anti-phospho p38 (Thr180/Tyr182) (2BB10, Cell Signaling), and anti-β-actin (C4, Millipore), anti-NKG2A (Abnova), anti-GATA-3 (D13C9, Cell Signaling), anti-phospho-GATA-3 (Ser308) (EPR18118, Abcam), and anti-Histone H3 (BioLegend).

Cells were lysed in RIPA buffer, and whole-cell extracts were quantified by the Bradford assay (Bio-Rad). For assessment of IL-1β secretion and caspase-1 release, culture supernatants were collected, mixed with a 1/10 volume of 100% (wt/vol) trichloroacetic acid, and incubated for 10 min at 4 °C. The precipitated protein samples or cell lysates were resolved by SDS/PAGE and transferred to PVDF membranes (Millipore). The membranes were then incubated with the indicated primary antibodies, followed by an HRP-conjugated secondary antibody. The immunoreactive bands were detected using the Western Lighting Plus-ECL system (PerkinElmer) or the Opti-ECL HRP reagent kit (BIOMAN).

Universal type I IFN was purchased from PBL biomedical laboratories (Piscataway, NJ). Antibodies against FLAG (Sigma-aldrich), Tubulin (Sigma-Aldrich), HA (Covance, Denver, PA & Santa Cruz), β-actin (Sigma-Aldrich), UBB+1 (Millipore), GFP (Invitrogen), FK1 (Enzo life Sciences) and ubiquitin (eBioscience) were purchased from the respective manufacturers. Proteins were electroblotted onto nitrocellulose membranes (HyBond, Amersham Biosciences Inc). Rabbit anti-mouse ISG15 polyclonal antibodies have been described previously (Malakhova et al., 2003). Armenian hamster ISG15 monoclonal antibody is a kind gift from Dr. Debra Lenschow (Washington University, MA). Fluorophore conjugated secondary antibodies (Li-Cor) or horseradish peroxidase (HRP) conjugated secondary antibodies were used for detection with Odyssey system (Li-Cor) or western lighting plus-ECL system (PerkinElmer), respectively.