Mouse anti glyceraldehyde 3 phosphate dehydrogenase gapdh

Adenine and perindopril erbumine (PE) were purchased from MedChemExpress (Shanghai, China). The primary antibodies included rabbit anti-fibronectin (FN), rabbit anti-type IV collagen (Col-IV) (Abcam, Cambridge, MA, U.S.A.), rabbit anti-SIRT3, mouse anti-optic atrophy 1 (OPA-1), rabbit anti-superoxide dismutase 1 (SOD1), rabbit anti-NADPH oxidase 2 (NOX2), rabbit anti-NOX4, mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China), rabbit anti-dynamin-related protein 1 (Drp-1), rabbit anti-SOD2, and rabbit anti-catalase (Cell Signaling Technology, Beverly, MA, U.S.A.). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Life Technologies (Carlsbad, CA, U.S.A.).

Ras activities were analyzed using the Ras Activation Assay Kit (Millipore, Billerica, MA, USA), according to the manufacturer’s instructions with some modifications. Active Ras was pulled down with Raf-1 Ras binding domain (RBD)-conjugated agarose beads (Millipore) at 4 °C for 1 h. The protein samples were boiled in Laemmli (Bio Rad, Hercules, CA, USA) for 5 min. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed, then the proteins were transferred to polyvinylidene fluoride membranes (Bio Rad). The membranes were treated with Block Ace (KAC, Kyoto, Japan) for 1.5 h and incubated overnight at 4 °C with mouse anti-Ras (1:500; Millipore) or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2500; Proteintech, Rosemont, IL, USA) antibodies. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies, and developed with EzWestLumi One (Atto, Tokyo, Japan). Chemiluminescent signals were acquired using the Fusion FX Imaging System (Vilber Lourmat, Marne La Vallée, France).