Nb100 106

IGHG3, IGHA1, S100A8, lactoferrin, and OGG1 were analyzed by Western blotting using a rabbit anti-human IGHG3 polyclonal (MBS248789; MyBioSource, San Diego, CA, USA), rabbit anti-human IGHA1 polyclonal (MBS9206028; MyBioSource), rabbit anti-human rat S100A8 polyclonal (MBS127619; MyBioSource), mouse anti-human lactoferrin monoclonal (ab10110; Abcam, Cambridge, UK), and rabbit anti-human OGG1 polyclonal (NB100-106; Novus Biologicals, Centennial, CO, USA) antibodies, respectively. The proteins were subjected to polyacrylamide gel electrophoresis using 10% (for IGHG3) or 15% (for IGHA1, S100/A8, lactoferrin, and OGG1) gel. The resolved proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated with secondary antibodies (goat anti-rabbit antibody A120-101P for IGHG3, IGHA1, S100A8, and OGG1 and goat anti-mouse antibody for lactoferrin; Bethyl Laboratories, Montgomery, TX, USA) diluted at 1:10,000 (IGHG3 and S100/A8) and 1:2000 (IGHA1, lactoferrin, and OGG). All analyses were performed in triplicate. The protein concentration was determined by measuring the optical density of the specific immunoreactive bands using Image J software (NIH, Bethesda, MD, USA).

After the HVL and LVL cell populations were sorted and collected, the cells were lysed in ice-cold Tris buffer (50 mM, pH 7.4) containing 1 mM DTT, 1 mM EDTA, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, phosphatase inhibitor, and protease inhibitor (Calbiochem. Millipore) for 30 min on ice and then centrifuged at 13,000 x g for 20 min. The protein concentration was determined using the Bio-Rad protein assay. A total of 10 μg of protein lysate was resolved by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked in 1X TBST containing 5% non-fat dried milk and then incubated with primary antibodies at 4°C overnight. The primary antibodies were against HCV NS3 (MAB8691, Merck Millipore, KGaA, Germany), HCV NS5A (MAB8694, Merck Millipore), HCV core (clone C7-50, MA1-080, Thermo Fisher, USA), CDK4 (clone DCS-31, C8218, Sigma-Aldrich), OGG1 (NB100-106, Novus Biologicals, USA), XPC, ATM (PA1-16503, Thermo Fisher), p-ATM (pSer1981, clone 10H11.E12, MA1-46069, Thermo Fisher), GAPDH (clone GAPDH-71.1, G8795, Sigma-Aldrich), and actin (clone AC-40, A3853, Sigma-Aldrich). The membranes were stained with HRP-conjugated secondary antibodies, and the signals were developed with chemiluminescence reagents (Amersham Biosciences, CA, USA). The chemiluminescent signal was captured by an ImageQuant™ LAS 4000 mini system (GE Healthcare Life Sciences).

Anti-RelA (MAB5078, R&D, Minneapolis, MN, USA) and anti-OGG1 (NB100-106, Novus) antibodies (Ab) were used in PLA with Duolink PLA kit (DUO 92010, Millipore Sigma, ThermoFisher Scientific, San Jose, CA, USA) according to the manufacturer’s instructions. The nuclei were counter-stained with DAPI. The PLA signals were visualized in a fluorescence microscope (ECHO) at 20× magnification.