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Superoxide dismutase was assayed spectrophotometrically at 414 nm by measuring the rate of pyrogallol auto-oxidation, as described by Marklund (1985). For determination of glutathione peroxidase, tissue was homogenized in cold buffer containing 50 mm Tris�CHCl, 5 mm EDTA, 1 mm dithiothreitol, pH 7.5, and centrifuged at 10,000g for 10 min (4��C). The supernatant was then processed using a cellular glutathione peroxidase assay kit (Calbiochem, Darmstatd, Germany). Absorbance was recorded at 340 nm for 3 min, considering that the rate of decrease in absorbance is directly proportional to glutathione peroxidase activity. Catalase activity was assayed spectrophotometrically at 240 nm by measuring the rate of consumption of hydrogen peroxide from 30 ��m hydrogen peroxide, at pH 7.4, in quartz cuvettes GPX4 of 1 cm path length following the method of Aebi (1984). Results are expressed as Katal units. Enzymatic antioxidant activities were normalized on the basis of protein content. Caspase-3-like activity was determined by measuring the cleavage of the substrate Ac-DEVD peptide conjugated to p-nitroaniline (pNA) with a colorimetric assay (Chemicon, Temecula, CA, USA; catalogue no. APT165). The assay is based on the spectrophotometric detection of the chromophore find more pNA after cleavage from the labelled substrate DEVD-pNA by caspases that recognize the DEVD sequence. Liver protein extract (50 ��g) was incubated with caspase substrate in the dark at 37��C. Chromogenic substrate pNA was cleaved by caspase-3, and absorbance was measured at 405 nm. Substrate cleavage is reported as the relative Osimertinib research buy activity compared with the control group. Data were normally distributed. Results are expressed as means �� SEM of eight animals. Data were evaluated using the GraphPad InStat statistical software (GraphPad Software, Inc., San Diego, CA, USA). The means of the experimental groups were analysed by two-way ANOVA. The Student�CNewman�CKeuls multiple comparison test was used to identify significant differences (when P