Horseradish peroxidase conjugated donkey anti rabbit igg

The tissues and cells were homogenized in the lysis buffer. After protein quantification, 40 μg of protein was loaded onto SDS-PAGE gels. Then, protein extracts were electrophoresed, blotted, and then, incubated with primary antibodies. The antibodies were detected using 1:10,000 horseradish peroxidase-conjugated donkey anti-rabbit IgG and donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA). Western blotting luminol reagent was used to visualize bands. The band intensities were quantitated by Image J software.

Viruses were allowed to bind to established human cell clones cultured in 12 well plates (2.0 × 10⁵ cells/well). Briefly, cells were washed twice with PBS (+), inoculated with viruses at an m.o.i. of 10, and then incubated at 4 °C for 1 h to prevent endocytosis. The cells were then washed five times with ice-cold PBS (+) and lysed in PBS (−) containing 2% SDS. The protein concentration of the resulting cell lysates was measured using the bicinchoninic acid protein quantification kit (Thermo Fisher Scientific). Next, 10 μg of protein per lane was subjected to electrophoresis in a 10% SDS-polyacrylamide gel. The proteins were then transferred to PVDF membranes and blocked overnight at 4 °C in PBS (−) containing 5% non-fat milk and 0.1% Tween 20. The membrane was then exposed for 1 h at room temperature to the aforementioned rabbit polyclonal antivirus antibody (diluted 1:1000 in PBS (−) containing 5% non-fat milk and 0.1% Tween 20), followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG (diluted 1:10,000 in PBS (−) containing 0.1% Tween 20; Jackson ImmunoResearch, West Grove, PA, USA). Between steps, the membranes were washed three times with PBS (−) containing 0.1% Tween 20. Reactive bands were visualized using a chemiluminescence system (Thermo Fisher Scientific) and Fuji XR film.

AKT2 rabbit mAb, PPARγ rabbit mAb, Acetyl‐CoA carboxylase rabbit mAb, and β‐actin mouse mAb were purchased from Cell Signaling Technology (Beverly, MA). Rabbit anti‐FAS and SREBP1 mouse mAb were from Abcam Inc (Cambridge, MA). Horseradish peroxidase‐conjugated, donkey anti‐Rabbit IgG and donkey anti‐Mouse IgG were purchased from Jackson ImmunoResearch (West Grove, PA).

Antibody recognizing precursor sterol regulatory element binding protein-1 (SREBP-1) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-fatty acid synthase (FAS) and antinuclear form of SREBP-1 antibodies were supplied by BD Biosciences (Franklin Lakes, NJ). Stearoyl-CoA desaturase-1 (SCD1), phospho-AMP-activated protein kinase (p-AMPK), and acetyl-CoA carboxylase (ACC) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody and T0901317 (T090) were supplied by Calbiochem (San Diego, CA, USA). Anti-β actin antibody, insulin, and glucose were purchased from Sigma-Aldrich (St. Louis, MO). Horseradish peroxidase-conjugated donkey anti-rabbit IgG, and alkaline phosphatase-conjugated donkey anti-mouse IgG were obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA). Eicosapentaenoic acid (EPA), docosahexaenoic acids (DHA), and compound A (CpdA) were supplied by Cayman Chemical (Ann Arbor, MI, USA). GW1100 and AMG-1638 were kindly donated from LG Chem Ltd. (Seoul, South Korea).