Cu znsod

Cells were harvested, washed two times with ice-cold PBS and then resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing 1% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, 5 µg/mL pepstatin A, 1 µg/mL chymostatin, 5 mM Na₃VO₄ and 5 mM NaF. The cell lysate was centrifuged at 13,000× g for 20 min at 4 °C. Protein concentration was determined using the BCA assay (Sigma, St Louis, MO, USA). Proteins were separated by Tris-Glycine SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with antibodies, indicated as follows: p-Akt and Akt (1:1000, Cell Signaling Technology, Beverly, MA, USA); p-ERK and ERK (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); mTOR (1:500, Santa Cruz Biotechnology); Bax and Bcl-2 (1:500, Santa Cruz Biotechnology); catalase, Cu/Zn-SOD, Mn-SOD (1:1000, Cell Signaling Technology), and GAPDH (1:1000, Assay Designs, Ann Arbor, MI, USA). The membrane was exposed to X-ray film; protein bands were scanned and measured using ImageJ analysis software (version 1.37; Wayne Rasband, NIH, Bethesda, MD, USA), and normalized by GAPDH, an internal control.

The proteins of the kidney tissues were extracted using a Pro-Prep Protein Extraction Kit (Intron Biotechnology, Inc., Seongnam, Korea) according to the manufacturer’s instructions. The protein concentration was measured using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Equal kidney protein samples were separated by 12 or 15% SDS-PAGE and transferred to nitrocellulose membranes. For immunodetection, the blots were incubated overnight at 4°C in PBS containing 0.1% Tween-20 and 5% skim milk with primary antibodies raised against the following proteins: AT1R (Santa Cruz Biotechnology, Santa Cruz, CA), Copper/zinc superoxide dismutase (Cu/ZnSOD) (Enzo Life Sciences, Inc., NY), Manganese-superoxide dismutase (MnSOD), catalase, type IV collagen, and SGLT2 (all from Abcam); β-actin (Sigma-Aldrich); The second antibody was HRP-linked anti-Rabbit IgG (Cell Signaling Technology, Beverly, MA) for AT1R, Cu/ZnSOD, SGLT2, and HPR-linked anti-Mouse IgG (Cell Signaling Technology) for MnSOD and β-actin. The protein bands were detected using a chemiluminescence imaging Systems (Fusion SL4-3500, Viber-Lourmat, France), and band densities were measured by Quantity One software (Bio-Rad).

Cells were lysed with Radioimmunoprecipitation assay buffer (RIPA) (Sigma-Aldrich) supplemented with protease inhibitors (Roche Complete protease inhibitor cocktail, Roche, Basel, Switzerland). Equal amount of lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). Membranes were blocked with 5% w/v milk (Blocking grade milk, Bio-Rad) for 1 h and incubated overnight at 4^oC with primary antibodies against gelsolin (Abcam, UK), Cu/ZnSOD (Cell Signaling, MA, USA), MnSOD (BD), VDAC (Cell Signaling); GAPDH (BD Biosciences) and β-actin (Sigma-Aldrich). The membranes were washed and incubated with horse radish peroxidase-conjugated secondary antibodies. Signals were visualized using chemiluminescence substrate (Thermo Scientific, MA, USA).