Rabbit anti denv ns3

U2OS cells were transfected with the indicated plasmids using XtremeGene360 (Roche). When specified, cells were infected with DENV for the indicated timepoints. Cells were lysed in 1x RIPA + protease inhibitor + 0.1% SDS cocktail (Sigma). Lysates were sonicated prior to being separated by SDS PAGE using a 4-20% Tris-glycine polyacrylamide pre-cast gel (BioRad) and transferred to nitrocellulose membranes. Following 30 min of blocking in PBS + 10% non-fat milk, membranes were probed with the indicated primary antibodies: mouse anti-V5 (Invitrogen), rabbit anti-GFP (Proteintech), mouse anti-GFP (Proteintech), mouse anti-actin (Proteintech, rabbit anti-actin (Proteintech), and rabbit anti-DENV NS3 (GeneTex) followed by near-infrared dye-conjugated secondary antibodies (LiCor) diluted in PBST + 5% non-fat milk and imaged on an Odyssey CLx imaging system (LiCor).

Cells were lysed in a modified radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 0.5% sodium deoxycholate, and 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail II (Millipore, Burlington, MA, USA), and post-nuclear lysates were harvested by centrifugation. Quantified protein (between 5 and 15 µg) was added to a 4× SDS protein sample buffer (40% glycerol, 240 mM Tris-HCl (pH 6.8), 8% SDS, 0.04% bromophenol blue, 5% beta-mercaptoethanol), resolved by SDS/PAGE, transferred to nitrocellulose membranes in a 25 mM Tris-192 mM glycine-0.01% SDS buffer, and blocked in StartingBlock buffer (Thermo-Fisher). Membranes were incubated with primary antibodies for 2 h at room temperature. After washing with PBS-T buffer (1× PBS, 0.05% Tween 20), membranes were incubated with species-specific horseradish peroxidase-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA, 1:5000) followed by treatment of the membrane with Clarity enhanced chemiluminescence (ECL) (BioRad) and imaging on X-ray film. The following antibodies were used for immunoblot: rabbit anti-GFP (Thermo-Fisher, 1:5000), rabbit anti-ZIKV NS3 (GeneTex, Irvine, CA, USA 1:5000), rabbit anti-DENV NS3 (GeneTex, 1:5000), anti-FLAG M2 (Sigma, 1:5000), and mouse anti-Tubulin (Sigma, 1:5000).