Viperin antibody

For frozen tissue, lysates were prepared using a RIPA lysis buffer (50mM Tris (pH7.4), 150mM NaCl, 2mM EDTA, 1% Nonidet P-40, 50mM NaF, 0.1% SDS and 0.5% sodium deoxycholate with PhosStop Phosphatase-Inhibitor Cocktail tablet and protease-inhibitor cocktail tablet (Roche). The homogenate was centrifuged at 4°C for 10 min at 14,000g and the supernatant was used for western blot. The protein content of the supernatant was quantified using bicinchoninic acid reagents and BSA standards. Equal amounts of protein were separated using a polyacrylamide SDS-PAGE gel. After SDS-PAGE, proteins were transferred to PVDF membrane. The membrane was blocked for 1 h at room temperature followed by incubation overnight at 4°C with viperin antibody (Abcam), phospho-PKCμ, phospho-ACC, phospho-Akt, or GAPDH antibodies (Cell Signaling). After overnight incubation, the blots were incubated with HRP- conjugated secondary antibodies at a dilution of 1:5,000 for 1 h at room temperature. Bands were visualized by ECL plus (Thermo Scientific) according to the manufacturer's instructions and quantified using Image lab software.

The following reagents were purchased from the indicated suppliers: Ham’s F-12 medium, Eagle’s minimal essential medium, Hank’s balanced salt solution (HBSS), penicillin-streptomycin-amphotericin B, L-glutamine, TRIzol reagent, sodium pyruvate, nonessential amino acids, gentamicin, fetal bovine serum (FBS), dNTPs, oligo(dT), random hexamers, and Superscript III from Invitrogen Life Technologies (Burlington, Ontario, Canada). Bronchial epithelial cell growth medium (BEGM) was from BioWhittaker (Walkersville, MD). Gene expression kits for GAPDH, IL-6, CXCL5, and HERC5 were from Applied Biosystems (Foster City, CA); antibody pairs for CCL5, CXCL5, and CXCL10, as well as recombinant standards for each assay were from R&D Systems (Minneapolis, MN); TaqMan master mix and complete protease inhibitor cocktail tablets were from Roche Diagnostics (Laval, Quebec, Canada); western blot antibody for IRF7 was from BioLegend (San Diego, CA), while the viperin antibody was from Abcam (Toronto, ON). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).