Western Blot Analysis of Kidney Proteins

The protein expression was determined using Western blot analysis as described [16 (link)]. Briefly, the kidney tissue and cell lysates were separated on SDS-polyacrylamide gel electrophoresis followed by transfer to polyvinylidene difluoride membranes. Then, the membranes were incubated with various antibodies including anti-p-eIF2α (1 : 5000, Cell Signaling Technology), anti-p-IRE1α (1 : 4000, Santa Cruz Biotechnology), ATF6α (1 : 5000, Cell Signaling Technology), anti-pJNK (1 : 5000, Cell Signaling Technology), anti-CHOP (1 : 1000, Santa Cruz Biotechnology), anti-pSrc (1 : 1000; Cell Signaling Technology), anti-pFyn (1 : 1000; Santa Cruz Biotechnology), and anti-β-actin (1 : 1000) overnight at 4°C on a shaker. Then, the membranes were incubated with respective secondary antibodies, washed, and reacted with an enhanced chemiluminescent sensitive plus reaction (BioFX Laboratories, Inc., Owings Mills, MD, USA). The bands were quantified by using ImageJ software and normalized by β-actin.