Anti cyp4a

Homogenates from the frozen renal cortex were prepared in 500 µL lysis buffer containing 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 150 mM sodium chloride, 50 mM Tris-hydrochloride, 100 mM EDTA, 1% Tergitol (NP40), 100 mM PMSF, 1× of protease inhibitor cocktail containing aprotinin and leupeptin and phosphatase inhibitors cocktail (Bio-world). Homogenates were incubated for 1 h at 4 °C and centrifuged at 10,000× g for 30 min at 4 °C. Proteins concentrations in the supernatants were measured using the Lowry quantification method (Bio-rad Laboratory, Hercules, CA, USA). For immunoblotting, proteins (40 μg) were separated on 12.5% polyacrylamide SDS-gel electrophoresis and transferred to nitrocellulose membranes. Blots were incubated with rabbit anti-CYP4A (1:500; Abcam, Cambridge, UK), and Mouse HSC-70 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The primary antibodies were detected using horseradish peroxidase-conjugated IgG. Enhanced chemiluminescence helped in visualizing the bands. Densitometric analysis was performed using Image J software (1.53 e, U.S. National Institutes of Health, Bethesda, MD, USA).