Goat anti rabbit igg hrp secondary antibody sc 2004

Western blotting was performed according to methods previously described^{22 (link)}. Briefly, 50 ug of total lysate from kidney tissue were subjected to 15% polyacrylamide gel electrophoresis and transferred to cellulose acetate membranes. The membranes were blocked with 1× casein solution for approximately 4 h and then incubated with rabbit monoclonal anti-P-AMPK (Ser485) (2537), anti-P-Akt (Ser473) (4058), anti-Akt (4685), anti-cleaved caspase-3 (Asp175) (9664) and anti-Cleaved Caspase-9 (Asp330) (7237), all from Cell signaling technology; or rabbit polyclonal anti-Nrf2 (sc-722), anti-GAPDH (H-12) (sc-166574) and mouse monoclonal anti-HO-1 (sc-390991) anti-AMPK (sc-398861), all from Santa Cruz Biotechnology; or PI3K (ab86714) and p-PI3K (ab182651), both from Abcam Biotechnology, in blocking buffer for 2 h at room temperature. Membranes were then washed three times with Tris-buffered saline-Tween-20 (TBST), incubated with goat anti-rabbit IgG-HRP secondary antibody (sc-2004, Santa Cruz Biotechnology) and imaged by ChemiDoc XRS + Molecular Imager (Bio-Rad, USA). All images were using the free image analyzing software Image J 1.42 (n = 10 per group).

Samples were loaded onto 4-20% Criterion or Mini-PROTEAN TGX gels (Bio-Rad), transferred to PVDF membranes (Trans-Blot, Bio-Rad), blocked with 5% milk in Tris-buffered saline containing 0.05% Tween 20 (TBST), and probed with rabbit anti-GPX2 (ab137431, Abcam; 1 : 1000 in 5% milk in TBST) followed by a goat anti-rabbit IgG-HRP secondary antibody (sc-2004; Santa Cruz Biotechnology; 1 : 5000 in TBST). Membranes were developed using Clarity ECL Substrate (Bio-Rad) and imaged using a ChemiDoc System (Bio-Rad). For loading control, membranes were reprobed with a mouse anti-β-actin antibody (sc-47778, Santa Cruz; 1 : 1000 in milk in TBST) followed by an anti-mouse IgG-HRP secondary antibody (HAF007; R&D Systems; 1 : 5000 in TBST). Pixel density of GPX2 was normalized to the respective actin band density.