Ab97445

Western blotting was performed as follows. First, extraction Kit (Epigentek) was utilized to isolate protein samples. Thereafter, 8% SDS‐PAGE gel was employed to separate proteins, which were then transferred onto PVDF membranes (Millipore). Subsequently, the membranes were blocked with phosphate‐buffered saline with Tween‐20 (PBST) solution (Sigma‐Aldrich) and 5% bovine serum albumin (Sigma‑Aldrich; Merck KGaA) for 1 hour at room temperature. Subsequently, PVDF membranes were incubated with the primary antibody at 4°C overnight, including anti‐ME1 (1:1000, ab97445, Abcam), anti‐ME2 (1:2000, ab139686, Abcam), and anti‐MDH1 (1:1000, ab181091, Abcam), with β‐actin (1:200, ab115777, Abcam) being as the loading control. HRP‐conjugated anti‐rabbit antibody (GE Healthcare) was used as secondary antibody. The protein bands were visualized by LAS‐300 imaging system (Fujifilm).

After washed with PBS, cells were then lysed with the mixture of RIPA buffer (Invitrogen) and protease inhibitor. And the supernatant was collected after centrifugation. A bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) was used to calculate the protein concentration of each sample. Equal amount of proteins was separated on a SDS‐polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5% skim milk for 1 hr at room temperature and then incubated in the primary antibodies: rabbit anti‐human HIF‐2α antibody (1:1000, #NB100‐105, Novus, Colorado, USA), mouse anti‐human GOT1 (1:1000, #60317‐1‐Ig, proteintech, Rosemont, USA), rabbit anti‐human GOT2 antibody (1:1000, #14800‐1‐AP, proteintech, Rosemont, USA), rabbit anti‐human GLS1 antibody (1:600, #12855‐1‐AP, proteintech, Rosemont, USA), rabbit anti‐human GLUD1 antibody (1:1000, #ab168352, Abcam, Cambridge, MA), rabbit anti‐human ME1 antibody(1:1000, #ab97445, Abcam, Cambrigde, MA), mouse anti‐human MDH1 antibody(1:500, #ab76616, Cambrigde, MA) rabbit anti‐human β‐actin antibody (1:1000, #ab18162, Abcam, MA) at 4°C overnight. Horseradish peroxidase‐conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) and an ECL chemiluminescence kit (Pierce) were used to detect bound antibody.

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.