Ab76959

Protein extraction was performed in Radioimmunoprecipitation assay buffer (RIPA) (Auragene, Changsha, China) and centrifuged at 13000 rpm for 20 min at 4 °C. Protein concentrations were determined using a BCA protein assay kit (Auragene, Changsha, China). Proteins were separated on 10% SDS-PAGE gels and then blotted onto nitrocellulose membranes, and probed with Anti-Girdin antibodies (1:1000; ab179481, ABcam); Anti-Glut1 antibodies (1:1000; ab12939, ABcam); Anti-LDHA antibodies (1:2000; ab125683, ABcam); Anti-Hexokinase II antibodies (1:1000; ab76959, ABcam); Anti-HIF1α antibodies (1:2000; ab65069, ABcam); Akt polyclonal antibodies (1:2000; YT0175, Immunoway); Phospho-Akt (T308) polyclonal antibodies (1:2000; YP0007, Immunoway); and Anti-PI3 Kinase p85 antibodies (1:1000; 4292, Cell signaling), followed by the appropriate horseradish peroxidase-conjugated secondary antibodies (Auragene, Changsha, China). Immunodetection was accomplished via the ECL plus western blotting detection system (Auragene, Changsha, China). The signal intensity was determined using the IPP6.0 software. Immunoblotting with anti-β-actin antibodies (1:500) was performed as an internal control.

Cells were lysed in 1 × SDS sample buffer. Kidney tissue was homogenized by a polytron homogenizer (Brinkmann Instruments) in RIPA lysis buffer on ice. The supernatants were collected after centrifugation at 13,000 × g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay. An equal amount of protein was loaded into 10% or 15% wt/vol. SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-PPARα (ab24509, Abcam, Cambridge, MA, USA), anti-CPT1α (ab128568, Abcam), anti-ACADL (ab196655, Abcam), anti-HK2 (ab76959, Abcam), anti-LDH (3558, Cell Signaling Technology, Danvers, MA, USA), anti-PDK1 (ab110025, Abcam), anti-HIF-1α (ab1, Abcam), anti-PHD2 (4835, Cell Signaling Technology), and anti-tubulin (T6074, Sigma Aldrich). Western blot was performed at least three times independently. Chemiluminescence is applied for detecting proteins on western blot membranes. The enhanced chemiluminescent ECL substrate (32209, Thermofisher Scientific, Carlsbad, CA, USA) enables immunodetection of horseradish peroxidase (HRP)-conjugated secondary antibodies using an imaging system. Quantification was performed by measurement of the intensity of the signals with the aid of Image J software package.