Actin clone ac 40

Total protein was extracted using RIPA protein lysis buffer (cat. no. 89900, Thermo Fisher, USA) with freshly added 1% protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice and then centrifuged at 13,000× g for 20 min. A total of 10 μg of protein was determined using the Bio-Rad protein assay and used for Western blotting. After Western blotting, the samples were separated using SDS-PAGE and transferred to PVDF membranes. PVDF membrane blots were blocked with 5% skim milk, followed by incubation with primary antibodies for HCV NS3 (MAB8691, Merck Millipore, KGaA, Germany), SRC (AHO0051, Thermo Fisher, USA), and actin (clone AC-40, A3853, Sigma-Aldrich) overnight at 4 °C in blocking buffer. Finally, the membrane blots were conjugated with horseradish peroxidase (HRP) secondary antibodies for 2 h and the signals were developed with chemiluminescence reagents (Amersham Biosciences, CA, USA). Chemiluminescence-associated bands were identified using ECL (Bio-Rad, ChemiDoc XRS + System) and quantified.

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).

Total cellular protein was extracted using RIPA protein lysis buffer (cat. no. 89900, Thermo Fisher) with freshly added 1% protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min on ice and then centrifuged at 13,000 × g for 20 min. A Bicinchoninic acid (BCA) assay protein kit (Beyotime, China) was used to detect protein concentration. A total of 10 μg of protein was determined using the Bio-Rad protein assay and used for Western blotting. After Western blotting, the samples were separated using 10% SDS-PAGE and transferred to PVDF membranes. PVDF membrane blots were blocked with 5% skim milk, followed by incubation with primary antibodies for E-cadherin (610182, BD Biosciences), N-cadherin (610920, BD Biosciences), Vimentin (550513, BD Biosciences), and actin (clone AC-40, A3853, Sigma-Aldrich) overnight at 4 °C in blocking buffer. Finally, the membrane blots were conjugated with horseradish peroxidase (HRP) secondary antibodies for 2 h and the signals were developed with chemiluminescence reagents (Amersham Biosciences). Chemiluminescence-associated bands were identified using ECL (Bio-Rad, ChemiDoc XRS + System) and quantified.