Ab157098

The lysate obtained from 1 × 10⁶ cells as previously reported [26 (link)] was subjected to a Bradford assay (Pierce™ Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific) to determine protein concentration using Bovine Serum Albumin as standard. Thirty micrograms of proteins were resolved by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked with a tris-buffered saline solution containing 5% non-fat dry milk and 0.5% Tween for 1 h at room temperature. Subsequently, membranes were probed overnight at 4 °C with anti-ACLY (ab157098, Abcam, Cambridge, MA), anti-ME1 (ab97445, Abcam), anti-NF-κB/p65 (ab16502, Abcam) or anti-β-actin (ab8227, Abcam) primary antibodies. The membranes were then incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The immunoreactions were detected by WesternBright™ ECL (Advansta, Menlo Park, CA, USA) at Chemidoc™ XRS detection system (Bio-Rad Laboratories). Image Lab Software (Bio-Rad Laboratories) was used for image acquisition and densitometric analysis.

Cell lysates were harvested and subjected to immunoblot analysis as previously described [24 (link)]. The method is based on SDS-PAGE followed by protein transfer to nitrocellulose membrane and treatment with specific antibodies. It is a widely used method with a remarkable reproducibility. The following specific antibodies against ATP citrate lyase (ab157098, Abcam, Cambridge, MA, USA) or anti-β-actin (ab8227, Abcam) were used. After 1-h incubation with Goat anti-Rabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the immunoreaction was detected by using the WesternBright ECL horseradish peroxidase substrate (Advansta, Menlo Park, CA, USA) in a ChemiDoc XRS Detection System equipped with the Image Lab Software version 5.2.1 (Bio-Rad Laboratories, Hercules, CA, USA) for image acquisition and densitometric analysis. Representative western blots from three independent experiments with similar results are shown in the Figures.