Pierce bca protein assay reagent a

The cells on the plate were washed with ice cold PBS twice and lysed with radioimmunoprecipitation assay buffer (RIPA buffer). Tumor tissues were washed with ice cold PBS and 20 mg of each sample were homogenized with a homogenizer on ice. After centrifugation at 12,000 rpm, 4°C to remove the debris, protein concentration was measured with Pierce BCA Protein Assay Reagent A (Thermo Fisher Scientific US, Cat# 23223). Approximately 25 μg (from cells) or 50 μg (from tissue) of protein was loaded into each well and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in a 10% polyacrylamide gel and transferred to immunoblot nitrocellulose (NC) membranes (Millipore). The membranes were incubated with primary antibody against YWHAZ (Abcam, Cat# ab51129, 1:500 dilution) or GAPDH (Cell Signaling Technology, Cat# 5174, 1:1000 dilution), followed by incubation with horseradish peroxidase conjugated secondary antibody. ECL Western blot analysis was used to detect the binding of primary and secondary antibodies in the Tanon 5200CE Chemi-image system (Abclonal, Woburn MA, USA).

J774A.1 cells (0.5 × 10⁶ cells/well in 24-well plates) were treated with HOSCN or co-treatments of HOCl with SCN⁻ as described above. To assess whether thiol oxidation could be reversed, total cellular thiols were measured after 1 h treatment and following re-incubation in DMEM for 2, 4 or 24 h. The cellular thiol concentrations were assessed using the ThioGlo-1 assay, as described previously, with fluorescence measured at λ_ex 384 nm and λ_em 513 nm [46 (link)]. Thiol concentrations were quantified using a standard curve constructed with GSH. Total protein concentrations were quantified by BCA assay (Pierce™ BCA Protein Assay Reagent A, ThermoFisher) assay to normalize thiol concentrations, as previously [41 (link)].