Anti idh1

Whole‐cell extracts were obtained using RIPA buffer composed of 50 mM Tris–HCl (pH 7.5), 15 mM NaCl, 1% NP‐40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitors. In the Western blotting analysis, 30 μg of total protein was loaded onto a 12.5% sodium dodecyl sulfate‐polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was blocked at 4°C overnight in TBS containing 5% Phospho Blocker Blocking Reagent and 0.2% Tween‐20 and then incubated at 4°C overnight with the following primary antibodies (Abs): anti‐IDH2 (1:500 dilution), anti‐Hif‐1α (1:200 dilution, Santa Cruz Biotechnologies, Dallas, TX, USA), anti‐PHD2 (1:500 dilution, Invitrogen, Waltham, MA, USA), anti‐GLUT1 (1:200 dilution), anti‐TIGAR (1:1,000 dilution), anti‐anti‐TKT (1:2,500 dilution), anti‐CTPS1 (1:1,000 dilution), anti‐PKM2 (1:1,000 dilution), anti‐LDHA (1:2,000 dilution), anti‐IDH1 (1:500 dilution; Abcam, Tokyo, Japan), anti‐ME1 (1:500 dilution), and anti‐G6PD (1:100 dilution). The blots were incubated with a peroxidase‐labeled secondary Ab for 1 h. After PBS washing, the signals were detected using enhanced chemiluminescence reagents with the ECL plus Western Blotting Detection System and analyzed using the LAS 3000 system (GE Healthcare, Los Angeles, CA, USA).

The method to prepare cell lysate was the same as in 2.9. The lysates were diluted by 5 × loading buffer (Thermo fisher, cat. no. 39001) followed by boiling for 10 min. 50 μg protein was loaded onto the 10% SDS-PAGE gels and was separated. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes and the membrane was blocked with 5% non-fat milk (w/v) at room temperature for 1 h followed with primary antibody incubation overnight at 4 °C. The primary antibodies used here include: anti-IDH1 (1:1000, Abcam, cat. no. ab172964), anti-ME1 (1:2000, Abcam, cat. no. ab97445), anti-MDH1 (1:10000, Abcam, cat. no. ab180152), anti-Visfatin (1:1000, Abcam, cat. no. ab236874), anti-LDHA (1:1000, Cell Signal Technology, cat. no. 2012), anti-LDHB (1:200, Santa Cruz Biotechnology, cat. no. sc-100775), anti-beta actin (1:10000, Abcam, cat. no. ab8226), anti-GAPDH (1:10000, Proteintech, cat. no. 60004-1-Ig). The membrane was washed with TBST for three times, and then incubated with HRP-conjugated secondary antibody (1:10000, BBI, cat. no. D110058 and D110087) at room temperature for 1 h. The membrane was washed with TBST for three times and protein bands were detected by adding ECL (PerkinElmer, cat. no. NEL103001EA) and captured on a Chemiluminescent Imaging System (Tanon, Shanghai, China).