Image station 2000 mm

Aliquots of heart tissue (50 mg) were homogenized in liquid nitrogen and dissolved in lysis buffer. Protein concentrations were determined by BCA protein quantitative assay. The protein lysates were loaded onto 10% SDS-polyacrylamide gel for separation, electrotransferred to PVDF membranes, and blocked in 5% nonfat milk in Tris-buffered saline, Membranes were incubated overnight using primary antibodies, (caspase-3 (Cell Signaling Technology Inc., no. 9662, USA) diluted to 1 : 1000, p-53 (Bioworld Technology Inc., BS3736, Louis Park, USA) diluted to 1 : 500, fas (Assay Designs, ADI-AAP-221D, USA) diluted to 1 : 500, Bcl-2 (Cell Signaling Technology, no. 2876, USA) diluted to 1 : 1000, and Bax (Cell Signaling Technology, no. 2772, USA) diluted to 1 : 1000) at 4°C. This step was followed by secondary antibodies, which were conjugated using horseradish peroxidase. We performed enhanced chemiluminescence (Merck-Millipore, Germany) detection. The images were captured and documented using a CCD system (image station 2000MM, Kodak, Rochester, NY, USA). Quantitative analysis of these images was performed using Molecular Imaging Software Version 4.0, which was provided by Kodak 2000MM System. The optical density was normalized against actin [10 (link)].

Liver samples were lysed using lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100) supplemented with protease inhibitors (1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin, and 50 μg/mL polymethyl sulfonyl fluoride). Equal amounts of tissue lysate were resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were then transferred onto polyvinylidene fluoride membranes (16916600, Roche), blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h at room temperature, and probed with primary antibodies against acyl-coenzyme A oxidase 1 (ACOX1) (AB091), long-chain fatty acid CoA ligase 1 (ACSL1) (A1000), short-chain acyl-CoA dehydrogenase (ACADS) (A0945), carnitine palmitoyl transferase 1 (CPT1) (A5307), and β-actin (A026) (all from ABclonal Technology). The specific proteins were detected using horseradish peroxidase-conjugated secondary antibodies (sc-2004, Santa Cruz Biotechnology), developed with SuperSignal West Pico Chemiluminescent Substrate (34,080, Thermo Scientific), and visualized using a Kodak Image Station 2000 MM. Immunoblotting results were quantified using Image J software (1.49 s). All the immunoblotting assays were performed using three biologic replicates.

The cells were treated with the lysis buffer (Cell Signaling Technology), and the protein extracts were dissolved in a sample buffer containing 50 mM Tris-HCl, 2% SDS, 10% glycerol, and 100 mM dithiothreitol (pH = 6.80). Proteins were separated using SDS-PAGE in 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were performed with anti-BMP-2 antibody, anti-VEGF antibody, anti-ALP antibody (Abcam), anti-β-catenin antibodies, anti-GSK-3β antibodies, and anti-C-myc antibodies (Santa Cruz Biotechnology, CA). The bands were captured and documented using a CCD system (Image Station 2000 MM, Kodak, Rochester, NY, USA). The blots were stripped and reprobed with anti-actin antibodies to demonstrate equal loading and to enable between-group protein content normalization. Densitometry of the bands was performed using Molecular Imaging Software Version 4.0 (Kodak).

Cells were lysed in RIPA Buffer (Sigma-Aldrich) and protein concentration was determined by using the BCA assay kit (Sigma-Aldrich). Total protein (40 μg) was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% or 5% gel and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were then incubated in 5% nonfat dry milk in Tris-buffered saline (TBS, pH 7.6) and then overnight at 4 °C with a rabbit monoclonal anti-DHC2 (#ab122525; Abcam) at a dilution of 1:1000, anti-KIF2B (#ab98214; Abcam) at a dilution of 1:2000 or anti-Phospho-H2A.X (#9718; Cell Signaling Technology) at a dilution of 1:1000. Membranes were then incubated at 37 °C for 1 h with an HRP-conjugated anti-rabbit IgG antibody (#7074; Cell Signaling Technology) at a dilution of 1:2000. After three washes with TBS, membranes were briefly incubated with chemiluminescent HRP substrate (#WBKLS0100; Millipore) and were photo-developed in Image Station 2000 MM (Kodak, Rochester, Minnesota, USA). Quantity One 4.6.2 (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify the density of the target protein in the membranes.

Total protein was extracted using cell lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100). Equal amounts of cell lysates were separated by 10% SDS-PAGE. Proteins were then transferred onto PVDF membranes (16916600, Roche), blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h, and probed with primary antibodies against ADH1C (A8081, ABclonal Technology), ALDH2 (A1226, ABclonal Technology), ALDH1A1 (A1802, ABclonal Technology), and β-Actin (4967L, Cell Signaling Technology). The specific proteins were detected with HRP-conjugated secondary antibodies (sc-2004, Santa Cruz Biotechnology), developed with SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Scientific) and visualized by Kodak Image Station 2000MM. Western blot results were quantified using Image J software.

HepG2 cells were lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with protease inhibitors (1 μg/mL Leupeptin, 1 μg/mL Aprotinin, 1 μg/mL Pepstatin, and 50 μg/mL PMSF). Equal amounts of cell lysates were resolved by 10% SDS-PAGE. Proteins were then transferred onto PVDF membranes (16916600, Roche), blocked with 5% non-fat milk in Tris-buffered saline containing 0.05% Tween 20 for 1 hour, and probed with primary antibodies against ADH1C (A8081, ABclonal Technology), ALDH1A1 (A1802, ABclonal Technology), ACSL1 (A1000, ABclonal Technology), ACOX1 (A8091, ABclonal Technology), ACADS (A0945, ABclonal Technology), and β-Actin (4967L, Cell Signaling Technology). The specific proteins were detected with HRP-conjugated secondary antibodies (sc-2004, Santa Cruz Biotechnology), developed with SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Scientific) and visualized by Kodak Image Station 2000 MM. Immunoblotting results were quantified using Image J software (1.49s). All the immunoblotting assays were performed in three biological replicates.

Twinkle helicase activity was observed by unwinding fluorescently labelled dsDNA substrates (both 5′ and 3′ overhangs) with increasing enzyme concentration. Reactions were carried out in buffer K (20 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 4 mM DTT, 3 mM ATP, 100 µg/ml BSA) for 30 minutes at 37 °C with 75 nM dsDNA probe followed by a 5-minute incubation with Proteinase K. The reaction mixtures were resolved in 8% native polyacrylamide gels in 0.5 × TBE buffer. The gels were scanned using a KODAK Image Station 2000MM.