Bca protein quantitation assay kit

For protein analysis, cells were lysed in RIPA buffer. The protein concentration was quantified with a BCA Protein Quantitation Assay Kit (Beyotime Biotech., Shanghai, China). Equal amounts of protein were loaded and separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore Corp., Billerica, MA, USA). The membranes were incubated overnight with appropriate primary antibodies at 4 °C. Bound antibodies were then visualized using horseradish peroxidase-conjugated secondary antibodies. A quantitative analysis was performed by using ImageJ software (U.S. National Institutes of Health). For the antibody information, the antibodies against CRY1, HK2 and PKM2 were purchased from Proteintech (Chicago, IL, USA). Anti-RORα were obtained from Santa Cruz Biotechnology (CA, USA). The antibodies against LC3II/I, BMAL1, ULK1 and ATG5 were purchased from Bioworld Technology, Inc. (Nanjing, China). The antibody against β-ACTIN was derived from Servicbio Technology Co., Ltd. (Wuhan, China). The secondary antibodies were obtained from Santa Cruz Biotechnology (CA, USA). Uncropped images are shown in Fig. S5.

Transfected and HG-induced HK-2 cells were spiked with RIPA lysis buffer (cat: P0013B, Beyotime, China), and the supernatant was collected after centrifugation to extract total protein. The protein concentration was analyzed with a BCA protein quantitation assay kit (cat: P0012, Beyotime, China), followed by denaturation by mixing the protein with 10% alkyl sodium sulfate buffer in a certain ratio and boiling for 5 min in a 95℃-water bath. Electrophoretic separation was performed using SDS-PAGE gels and transferred to 0.22 μm PVDF membranes (cat: ISEQ00010, Millipore, USA). Then, the membranes were blocked for 2 h at room temperature in 5% bovine serum albumin (BSA)(cat: A9647, Millipore, USA), followed by incubation with primary antibodies (KFL5 antibody, cat: 668,501-Ig, Proteintech, China; β-actin antibody, cat: 81115-1-RR, Proteintech, China) at 1:1000 dilutions overnight at 4℃. After washing with 0.05% tris buffered saline/Tween (TBST), the membrane was incubated with enzyme-labeled secondary antibody (Proteintech, China, 1:5000 dilution) for 2 h. Finally, proteins were displayed using the enhanced chemiluminescent luminescence (ECL) kit (cat: NEL103001EA, Perkin Elmer, USA) and protein band images were analyzed using the Bio-Rad ChemiDOC XRS system (Bio-Rad Corporation, USA). The original gel chart has been presented in the supplementary material.