Gpx assay kit

For the assessment of H₂O₂ content and antioxidant enzyme activities, the supernatant from fresh leaves was prepared according to Quan et al. [49 ] and used for the following measurement.
The content of H₂O₂ was analyzed using H₂O₂ Assay Kit (A064–1-1, Nanjing Jiancheng Bioengineering Institute, China) based on the manufacturer’s instructions. The measurement of POD, CAT and GPX activities was performed with Plant POD Assay Kit (A084–3-1, Nanjing Jiancheng Bioengineering Institute, China), CAT Assay Kit (A007–1-1, Nanjing Jiancheng Bioengineering Institute, China) and GPX Assay Kit (A005–1-2, Nanjing Jiancheng Bioengineering Institute, China), respectively, as the introduction described.

Wild-type seedlings and transgenic cotton seedlings were treated with 200 mM NaCl solution after four weeks. The concentration of glutathione peroxidase (GPX), peroxidase (POD), and catalase (CAT) in leaves was measured at various time intervals following salt treatment. After 0.5, 12 h, 48 h, and 72 h, five individual seedling leaves from each biological replication were collected, and examined for enzyme content. Control pots were irrigated with tap water. Weigh each centrifuge tube, add roughly 0.1 g leaves to each tube, and separate the treated cotton. The protein content of the crude leaf extract was measured before the enzyme activity content. Three separate biological replicates of each control and salt-treated sample (3–4 seedlings) were assessed. The CAT, POD and GPX activities were determined using a CAT Assay Kit (A007-1), POD Assay Kit (A084-3), and GPX Assay Kit (A004-3) according to the protocol manufactured by Nanjing Jiancheng Bioengineering Institute, Nanjing, China.

Pyrrole, 4-carbomethoxybenzaldehyde, propionic acid, MnCl₂·4H₂O, tetrahydrofuran (THF), ZrOCl₂·8H₂O, benzoic acid, DMF and 4'6-diamidino-2-phenylindole (DAPI) were purchased from Sinopharm Chemical Reagent Co. Ltd (Beijing, China). RPMI 1640 and fetal bovine serum (FBS) were purchased from Gibco RBL/Life Technologies (Grand Island, NY, USA). SOSG was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). 2'7'-DCFH-DA was purchased from Sigma-Aldrich Company (St Louis, MO, USA). GSH assay kit, GPX assay kit and LPO assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other regents were of analytical grade and used without any further purification.

The superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione peroxidase (GPX) activities were determined using a SOD Assay Kit (A001-1 Nanjing Jiancheng Bioengineering Institute, Nanjing, China), CAT Assay Kit (A007-1 Nanjing Jiancheng Bioengineering Institute, Nanjing, China), POD Assay Kit (A084-3 Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and GPX Assay Kit (A004-3 Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively, according to the manufacturer’s instructions.

Dulbecco’s modified Eagles medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibcol (Grand Island, NY, USA). MitoSOX™ Red Mitochondrial Superoxide Indicator was purchased from Molecular Probes Inc. (Eugene, OR, USA). All of the primary antibodies were obtained from Abcam (Cambridge, MA, USA). MDA, SOD, CAT, and GPx assay kits were obtained from Jiancheng Bioengineering Institute (Jiancheng, Nanjing, China). Other reagents were all purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.

GPA (purity >98%) was purchased from MedChemExpress (NJ, USA). MMPs, NF‐κB, GPX4 and Nrf2 signalling antibodies were obtained from CST (MA, USA). Recombinant human IL‐1β and ELISA kits were obtained from R&D systems (MN, USA). Griess reagent, MDA, GPX assay kits were acquired from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

The activities of SOD, CAT and GPx in the tissue homogenates were assessed using superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively, according to the manufacturer's protocols. SOD activity was based on the auto-oxidation of hydroxylamine, with the developed blue colour then measured at 550 nm. The decomposition of H₂O₂ by CAT was stopped by the addition of ammonium molybdate. The remaining H₂O₂ was then reacted with ammonium molybdate to generate a pale-yellow complex, which was measured at 405 nm. The activity of CAT was then calculated. A series of enzymatic reactions was activated by GPx in the homogenate, leading to the conversion of GSH (reduced glutathione) to oxidized glutathione (GSSG). The change in absorbance during this conversion was recorded spectrophotometrically at 412 nm (Shimadzu UV-2550, Japan). The TAC in tissue homogenates was detected using a total antioxidant capacity (TAC) assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).