Gsh px activity kit

Prob was obtained from Shanghai Aladdin Bio-chem Technology Co., Ltd (Shanghai, China). SOD, GSH-PX activity kits, MDA and ROS detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Keap1, Nrf2, SOD and GSH-PX antibodies were from Santa Cruz Biotechnology, Inc. (Texas, USA). Ho-1 antibody was from Merck Millipore (Massachusetts, USA). Other reagents were all provided from Sigma-Aldrich unless otherwise noted.

PNS was obtained from Yunnan Yunke Pharmaceutical Manufacture Co., Ltd. (Yunnan, China). Huperzine A (Hup A) was from Forward Group (Shanghai, China). The SOD, CAT, and GSH-PX activity kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Mouse 8-OHdG ELISA kit was from Vicmed Biotech Co., Ltd. (Xuzhou, China). SOD, CAT, GSH-PX, UCP4, and UCP5 antibodies were from Santa Cruz Biotechnology, Inc. (Texas, USA). β-Actin antibody was from Arigo biolaboratories (Hsinchu, China). Horseradish peroxide- (HRP-) labeled goat-anti-rabbit and goat-anti-mouse (for GSH-PX) IgG working solution and DAB staining solution were obtained from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). IRDye 800CW goat anti-mouse IgG and goat anti-rabbit IgG were from LI-COR Biotechnology, Inc. (Nebraska, USA). The ReverTra Ace® qPCR RT Kit was obtained from Toyobo Life Science Department (Osaka, Japan).

The antioxidant activity of ML1206 was measured in the presence and absence of oxidative stress in C. elegans. Synchronized to the L4 stage, nematodes were fed with ML1206 or E. coli OP50. After 48 h incubation, the nematodes were treated with or without 5 mM H₂O₂ for 2 h and washed with M9 buffer. Next, the nematodes were shaken for half an hour, washed three times to eliminate the influence of bacteria itself as much as possible, and centrifuged slightly. The nematodes were lysed using a rotor–stator on ice, accompanied by intermittent grinding (grinding 15 s, stopping 5 s) to prevent enzyme inactivation. The homogenate was centrifuged at 12,000 rpm, 4 °C for 3 min, and the supernatant was collected for enzymeatic activity determination. The T-AOC was determined using the Total Antioxidant Capacity Assay Kit (Beyotime, Shanghai, China), and each individual enzyme activity was determined using SOD, CAT, and GSH-PX activity kit (Nanjing JianCheng Bioengineering Institute, Nanjing, China) respectively. The protein content was determined by BCA Protein Assay Kit (Beyotime, Shanghai, China).