Anti pdhb

Total protein lysate was extracted by radioimmunoprecipitation assay (RIPA) lysis buffer with cOmplete protease inhibitor and PhosSTOP phosphatase inhibitor cocktails (both from Roche, Basel, Switzerland). PDHA, PDHB, PC, and β‐actin were separated by 10% v/v sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto PVDF membranes (GE Healthcare, Chicago, IL, USA). The protein expression of PDHA, PDHB and PC in different subclones was determined by western blotting using anti‐PDHA (Cell Signaling, #3205, 1:1000), anti‐PDHB (Abcam, Cambridge, UK; ab155996, 1:1000) and anti‐PC (Santa Cruz Biotechnology, Dallas, TX, USA; sc271493, 1:1000) antibodies, respectively.

The cell protein was extracted using RIPA buffer (Thermo). Protein concentration was determined by BCA Protein Assay (Beyotime). Total protein was loaded, fractionated by SDS-PAGE, transferred to PVDF membrane, and probed with anti-β actin (Abcam), anti-PKM (Abcam), anti-PFKM (Cell Signaling Technology), anti-PDHB (Abcam), and anti-IDH2 (Cell Signaling Technology). Signal was detected using Chemiluminescence imaging system (Tanon, Shanghai, China).

Renal clear cell carcinoma and adjacent noncancerous renal samples were obtained by radical nephrectomy from the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital) between 2005 and 2018. All diagnoses were confirmed by senior pathologists independently. Informed consent from all patients was acquired in the study. The study design and protocol were approved by the ethics committee of the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital). IHC assays were performed as previously described (49 (link), 50 (link)). Briefly, the primary antibody was diluted as follows: anti-PDHB (1:100, Abcam, USA).