Mouse anti hcv core

Cells were lysed in a modified radio immunoprecipitation assay (RIPA) buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100) supplemented with protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Millipore), and post-nuclear supernatants were harvested by centrifugation. Quantified protein was resolved by SDS/PAGE, transferred to PVDF membranes using the Turbo-transfer system (BioRad) and blocked with StartingBlock (Thermo-Fisher) or 3% bovine serum albumin (Sigma) in PBS with 0.1% Tween (PBS-T). Membranes were probed with specific antibodies, washed with PBS-T and incubated with species-specific HRP conjugated antibodies (Jackson ImmunoResearch, 1:5000), washed again with PBS-T, and treated with Pico PLUS enhanced chemiluminescent (ECL) reagent (Thermo-Fisher). The signal was then captured on X-ray film or by using a LICOR Odyssey FC. Antibodies used for immunoblot include mouse anti-HCV Core (1:250, Abcam), mouse anti-HCV NS3 (1:500, Abcam), rabbit anti-HCV NS4A (1:1000, Genscript [63 (link)]), mouse anti-HCV NS5A (1:500, 9e10, gift of Dr. Charles Rice), anti-Flag HRP (1:2500, Sigma), rabbit anti-HA (1:500, Sigma), and mouse anti-E1 (1:1000, A4, gift of Dr. Charles Rice).