2.8. Western Blot Analysis

The expression of HO-1, PINK1, Mfn1/2, OPA1, Drp1, Fis1, and β-actin in the rats' right kidney tissues was determined by Western blotting. The kidney tissue was lysed and clarified by centrifugation at 10,000 g for 10 min at 4°C. Equal amounts of soluble protein were subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Bio-Rad, USA). Membranes were blocked with 5% nonfat powdered milk in Tris-buffered saline and Tween 20 (TBST) and incubated further at 4°C overnight with primary antibodies for HO-1 (1 : 500) (10701-1-AP), Mfn1 (1 : 800) (ab104274), Mfn2 (1 : 800) (ab56889), OPA1 (1 : 800) (ab157457), Fis1 (1 : 500) (10956-1-AP), Drp1 (1 : 500) (ab184247), PINK1 (1 : 600) (23274-1-AP), caspase1 (1 : 800) (22915-1-AP), IL-1β (1 : 1,000) (ab9722), and β-actin (1 : 1,000) (TA-09). After being washed three times with TBST, the blots were incubated with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G (1 : 3,000) (ZB2301) at room temperature for 1 h. The area and integrated optical density of the bands were analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD).