Anti cox4i1

Whole kidney protein was extracted with lysis buffer. After centrifugation (13,000 rpm, 4 °C, 15 min), the lysate was mixed with 5× sample buffer and heated at 95 °C for 6 min. Total protein concentrations were measured using Bradford methods (BioRad Laboratories, Hercules, CA, USA). The lysate was subjected to SDS-PAGE gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were incubated overnight at 4 °C with the primary antibodies: anti-p-Src (1:1000; Cell Signaling Technology), anti-p-Fyn (1:1000; Santa Cruz Biotechnology), anti-PGC-1α (1:1000; Abcam), anti-TFAM (1:1000; Abcam), anti-NRF1 (1:1000; Abcam), anti-Cox4i1 (1:1000; Cusabio Biotech Co., Baltimore, MD, USA), anti-β-actin (1:1000), and anti-heat shock 70 kDa protein 8 (HSC70, 1:1000; Santa Cruz Biotechnology). The blots were reacted with peroxidase-conjugated secondary antibodies (Vector Laboratories, Inc., Burlingame, CA, USA), followed by an enhanced chemiluminescent sensitive plus reaction (BioFX Laboratories, Inc., Owings Mills, MD, USA). The positive immunoreactive protein bands were detected by LAS-3000 film (FUJIFILM Corporation, Tokyo, Japan). Each blot density was normalized to β-actin or heat shock 70 kDa protein 8 and compared with that of each control.