β tubulin

Cells and tissue samples were harvested and treated with lysis buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (Beyotime). Protein concentrations were determined using a BCA protein assay kit (Beyotime). Comparable amounts of proteins were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were probed with antibodies against PCK1 (1:1000; Cat# BS6870; Bioworld, Atlanta, GA, USA), TXNRD1 (1:500; Cat# 15140; Cell Signaling), or NRF2 (1:1000; Cat# 12721; Cell Signaling). Secondary antibodies conjugated to horseradish peroxidase were purchased from Abcam (Cambridge, MA, USA). Protein bands were visualized using Super Signal West Pico Chemiluminescent Substrate Kit (Millipore) and quantified using ImageJ software (National Institutes of Health, Bethesda, MA, USA, http://imagej.nih.gov/ij/). GAPDH (Cat# AF0006; Beyotime), β-actin (Cat# BL005B; Biosharp), laminB1 (Cat# AP6001; Bioworld), and β-tubulin (Cat# AP0064; Bioworld) were used as normalization controls. Nuclear and cytoplasmic extracts of cells were collected using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). All experiments were repeated three times independently.

Antibodies used in this study are listed as follows: DNA-PKcs (ab32566, Abcam, UK, 1:5000), phosphorylated DNA-PKcs (ab124918, Abcam, 1:5000), phosphorylated ATM (ab81292, Abcam, 1:5000), ATM (ab32420, Abcam, 1:5000), PI3KCA (ab124918, Abcam, 1:1000), phosphorylated histone H2AX (2577, Cell Signaling Technology, USA, 1:500 for IB, 1:100 for IHC), H2AX (7631, Cell Signaling Technology, 1:1000), IRF9 (76684, Cell Signaling Technology, 1:1000), eIF-2α (5324, Cell Signaling Technology, 1:1000), phosphorylated eIF-2α (3398, Cell Signaling Technology, 1:1000), JNK (9252, Cell Signaling Technology, 1:1000), phosphorylated JNK (9255, Cell Signaling Technology, 1:1000), PERK(3179, Cell Signaling Technology, 1:1000), CHOP (2895, Cell Signaling Technology, 1:1000), Ki-67 (9449 s, Cell Signaling Technology, 1:400), cleaved-Caspase-3 (9664 s, Cell Signaling Technology, 1:1000 for IB, 1:500 for IHC), Caspase-3 (9662, Cell Signaling Technology, 1:1000), CD4 (25229, Cell Signaling Technology, 1:200), CD8 (98941, Cell Signaling Technology, 1:400), GAPDH (AP0063, Bioworld, USA, 1:10000), α-tubulin (ARG65693, Arigo Biolaboratories, China, 1:5000), β-tubulin (AP0064, Bioworld, 1:10000), M1 E1 and NS3 (produced by Beijing Protein Innovation, China, 1:2000). Anticancer compounds used in this study were purchased from Selleckchem.

Protein was extracted from frozen liver samples weighing 50 mg, and the BCA Protein Assay kit (NO.23227, Thermos Scientific, Waltham, MA, USA) was used to measure protein concentrations following the instructions provided by the manufacturer. In total, 50 μg of protein was loaded per lane for electrophoresis on a 10% SDS-PAGE gel. After transferring the protein onto a nitrocellulose filter membrane, it was blocked in a 4% milk solution and incubated with primary antibodies (FASN, AB43451, 1:1000, AbSci, MD, USA; SREBP1, 14088-1-AP, 1:1000, ThermoFisher, Waltham, MA, USA) and secondary antibodies (111-095-003, Jackson ImmunoResearch, West Grove, PA, USA). For loading control, β-Tubulin (BS1699, Bioworld, Bloomington, IN, USA) was employed.

We prepared lysates and isolated proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred them onto polyvinylidene difluoride (PVDF) membranes. Blots were incubated with primary antibodies (anti-NLRP3 [AdipoGen Life Sciences, San Diego, CA, USA], anti–CASP-1 [R&D Systems, Inc., Minneapolis, MN, USA], anti–IL-1β [Santa Cruz Biotechnology, Dallas, TX, USA] or β-tubulin [bioWORLD, Dublin, OH, USA]) overnight at 4 °C. Incubation with secondary anti-goat–horseradish peroxidase (HRP) and anti-rabbit–HRP (Hangzhou MultiSciences [Lianke] Biotech Co., Ltd., Hangzhou, China) was performed at RT for 1 h. We used an ECL Plus Western Blot Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA) for antibody detection.

The implants attached with primary osteoblasts were washed with ice-cold PBS and lysed to release the whole proteins by RIPA buffer with 1 mM PMSF. The cell lysates were agitated at 4 °C for 30 min and then centrifuged at 4 °C for 20 min. The protein concentration was determined by the BCA assay. The protein extracts (30 μg per sample) were separated by 8% or 10% Tris-glycine SDS-PAGE and then transferred onto PVDF membranes (Millipore) after mixed with 5× loading buffer. The PVDF membranes were blocked in TBST (Tris Buffer Saline, 0.5% Tween-20) containing 5% BSA for 1 h and incubated overnight at 4 °C with primary antibodies to OCN (1:1000, Abcam, Cambridge, MA, USA), Runx2 (1:1000, Biorbyt Ltd., Cambridge, UK), Wnt3a (1:1000, Novus Biologicals, Littleton, CO, USA), Lrp6 (1:1000, Lifespan Bioscience, Seattle, WA, USA), β-catenin (1:1000, Millipore, Billerica, MA, USA), β-Tubulin (1:3000, Bioworld technology, Inc., Louis Park, MN, USA), and β-Actin (1:3000, Bioworld) in TBST containing 5% BSA. The membranes were then incubated with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 h at RT, and visualized by an ECL chemiluminescence system (GE ImageQuant 350, GE Healthcare). Semi-quantitative analysis was performed using the QuantityOne Software (Bio-Rad). β-Actin or β-Tubulin was used as an internal control for normalization.