Anti ldhb

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).