Ab179843

The U87 MG human glioma cells were seeded in 25 cm² flasks at the density 5 × 10⁵ cells. Cells were treated for 30 min with 200 µM H₂O₂ before lysis in radioimmunoprecipitation (RIPA) buffer (150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8). Western blot analysis was performed similarly as described in [15 (link)]. Oxidative stress defense cocktail (ab179843, Abcam, Cambridge, UK) was applied to estimate the expression level of catalase, superoxide dismutase 1, thioredoxin, and smooth muscle actin in cells. Anti-β-actin antibody (ab8227, Abcam, Cambridge, UK) was determined as a housing protein. The WesternBreeze chromogenic kit anti- rabbit was purchased from ThermoFisher Scientific. The image analysis of proteins on the membrane was performed with ImageJ software. Optical densities (O.D.) of the bands were detected with ImageJ and analyzed, the normalized O.D. values are the mean values from 4 measurements and are plotted in histograms (down). Error bars represent standard deviations. The level of significant difference was calculated with one-way ANOVA: * p < 0.05, ** p < 0.01.

For the Western blot experiments, cell pellets of SkMS/PCs were lysed in 8 M urea, 50 mM Tris–HCl, pH 8.0 with 1% SDS (1:1) containing protease inhibitor cocktail (Roche, Basel, Switzerland). The total protein concentration was determined using the Lowry method. A total of 50 μg of protein was separated on 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels and electrotransferred under standard conditions (30 min) using Trans-Blot Turbo to a PVDF membrane (all from Bio-Rad, Hercules, CA, USA). The membrane was soaked with blocking buffer containing non-fat milk (Bio-Rad, Hercules, CA, USA). Immunodetection was performed using: the anti-forkhead box O1 (FOXO1A) antibody −70 kDa (ab52857); anti-heat shock protein70 (HSP70) antibody 1:1000, 70 kDa (ab31010); anti-myoblast determination protein 1 (MYOD) antibody 1:1000, −50 kDa; anti-oxidative stress defense (Catalase- CAT 60 kDa superoxide dismutase- SOD1 16 kDa, thioredoxin- TRX 12 kDa, smooth muscle actin- ACT 42 kDa) antibodies cocktail (ab179843) 1:200; and anti-hypoxanthine-guanine phosphoribosyltransferase (HPRT) antibody 1:1000, 25 kDa (ab109021) (Abcam, Cambridge, UK). The detection of the target protein was achieved by incubating the membrane with Clarity ECL, Western Blot substrate and analyzed with ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).