Proteinruler 2

His-tagged recombinant LDHA and LDHB were expressed in E. coli and were purified by NTA nickel column (Qiagen, Hilden, Germany). The proteins were subjected to SDS-PAGE followed by Coomassie brilliant blue staining. The recombinant proteins were quantified based on the intensity of bands using the protein marker with known quantity (ProteinRuler II, TransGen Biotech, Beijing, China) as the reference. Next, the cell lysates were subjected to SDS-PAGE with quantified His-LDHA and LDHB and follow by WB using specific antibodies against LDHA and LDHB, respectively. Then the relative amount of endogenous LDHA and LDHB were quantified by comparing the intensity between His-tagged LDHA/B and endogenous LDHA/B based on the WB result using Image J. The relative expression levels of endogenous LDHA and LDHB in cell lines were then calculated according to the above results.

Bovine serum albumin (BSA), human serum albumin (HSA), Brilliant blue R–250, Pyronin Y, DAPI and SYPRO (R) Orange Protein Gel Stain were purchased from Sigma Chemicals Co. All other chemicals are of analytical reagent grade. JM109, DH10B, BL21(DE3) Chemically Competent Cell, ProteinRuler I and ProteinRuler II, were purchased from TransGen Biotech (Beijing, China). A Pierce Rapid Gold BCA Protein Assay Kit was purchased from Thermo Fisher Scientific. RecR, RuvA, RuvB, RecA and MutL were cloned into an expression vector, and the proteins were purified. Cell cultures were maintained in the DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 1% penicillin/streptomycin storage solution (100 U/ml penicillin, 100 mg/ml streptomycin), at 37°C in a humidified atmosphere containing 5% CO₂. All the solutions were prepared with sterile ultrapure water. The (Z)-1,2-dimethyl-4-(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (PyMDI) was synthesized according to the synthesis procedure described in [20 (link)].