Chemiluminescence reagent

Frozen RV was homogenized using ice-cold lysis buffer and proteinase inhibitor cocktail (Manchini et al., 2014 (link)). Lysates corresponding to 30 μg of protein were subjected to 10% SDS-PAGE. Separated proteins were transferred to PVDF membrane (Amersham Biosciences, NJ, USA) and transfer effectiveness was examined with 0.5% Ponceau S. After blocking with 5% non-fat dry milk for 2 h at room temperature, PVDF membranes were probed with Abcam (Cambridge, MA, USA) primary antibodies for rabbit ant-Akt₁ (1:5000), rabbit anti-p-Akt₁ (1:2500), rabbit anti-Caspase3; rabbit anti-Bax (1:1000), rabbit anti-Bcl-2 (1:1000), rabbit anti-Bcl-xL (1:500), rabbit anti-β-MHC (1:5000), rabbit anti-α-MHC (1:5000), rabbit anti-L-type Ca⁺⁺ (1:500), rabbit anti-ryanodine receptor (1:1000), rabbit anti-Serca 2 (1:1000), and rabbit anti-Na⁺/Ca⁺⁺ exchanger (1:100) in overnight incubation. Membranes were then washed five times with PBS and incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit (1:20,000; Zymed, CA, USA). Membranes were again washed five times with blocking buffer and then rinsed twice with PBS. Antibodies binding were detected by chemiluminescence reagents (Amersham Biosciences, NJ, USA), and images were captured using an Amersham Imager 600 system. Quantification of target proteins was normalized for the internal control glyceraldehyde 3-phosphate dehydrogenase.

The collected microglial samples were homogenized with Laemmli Sample buffer (BioRad), loaded onto 4–15% gradient Criterion gels (Bio-Rad) followed by electrophoretic transfer to Nitrocellulose membranes (Bio-Rad) using a standard protocol. Samples were normalized to 1 µg/ml and the loading volume was 20 µl/well. The membranes were incubated with rabbit polyclonal CD11b (2 µg/ml, Abcam) and mouse anti-β-actin (1∶5000, Sigma) over night at 4°C, followed by reactions with respective goat anti-rabbit (1∶5000) and –mouse (1∶5000) (Sigma) peroxidase conjugates. The protein bands for CD11b and β-actin were visualized by chemiluminescence reagents (Amersham, Piscataway, NJ) and quantified by Image J software.

Cells and tumor tissues were lysed in immunoprecipitation assay buffer (50 mM Tris-Cl [pH 7.4], 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 M phenylmethylsulfonyl fluoride, 10 µg each of aprotinin and leupeptin, and 1 mM Na3VO4). After centrifuged at 12,000 g for 30 min, the supernatant was collected and protein concentration was determined using the Lowry method. Equal amounts of protein were separated on 12% SDS-PAGE gels and blotted onto nitrocellulose membranes. The blots were incubated with anti-Prx1 (1∶5000, Upstate, USA) and anti-HO-1 (1∶2000, Abcam, USA) antibody. Antibody of β-actin (Sigma, St. Louis, MO) was used as a loading control. Immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced by chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ). The experiments were performed in triplicate.

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).

BP (C₁₂H₁₂O₂, 95%) was purchased from Lancaster Synthesis Ltd. (Newgate Morecambe, UK). Cisplatin, dimethyl sulfoxide (DMSO), [3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), crystal violet, DSD, Tween-20, methanol, and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies were all purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Polyvinyldifluoride (PVDF) membranes, BSA protein assay kit and chemiluminescence reagents were purchased from Amersham Biosciences (Arlington Heights, IL, USA).

BP (C₁₂H₁₂O₂, 95%) was purchased from Alfa Aesar (Ward Hill, NY, USA). Dimethyl sulfoxide (DMSO), crystal violet, [3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), Tween-20, methanol, and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The antibodies were all purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Polyvinyldenefluoride (PVDF) membranes, BSA protein assay kit and chemiluminescence reagents were purchased from Amersham Biosciences (Arlington Heights, IL, USA).