DPSCs expanded with or without H2O2 (0–200 μM) were maintained in T-75 flasks as above, until 80–90% confluent. Cultures were harvested with RIPA buffer (400 μL/flask, ThermoFisher Scientific), containing cOmplete™ Protease Inhibitor Cocktail (Roche), according to manufacturer’s instructions. Extracts were sonicated and protein concentration quantified (Pierce® BCA Protein Assay Kit, ThermoFisher Scientific). Protein samples (10 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on pre-formed 4–15% TGX™ gels (Mini-Protean® Tetra Cell System; Bio-Rad, Hemel Hempstead, UK); and electroblotting onto polyvinylidene difluoride membranes (Hybond™-P; ThermoFisher Scientific), using a Mini Trans-Blot® Electrophoretic Transfer Cell (Bio-Rad), per the manufacturer’s instructions. Membranes were blocked with 5% semi-skimmed milk/1% Tween 20 in Tris-buffered saline (TBS), for 1 h at room temperature. Membranes were immuno-probed with primary antibodies (Abcam, Cambridge, UK), specific to SOD1 (ab16831, 1:1000); SOD2 (ab13534, 1:1000); SOD3 (ab21974, 1:1000), and GSTZ1 (ab153995, 1:500). Normalized protein loading was confirmed by β-actin Loading Control (ab8227, 1:20,000, Abcam). Immuno-probing occurred in 5% semi-skimmed milk/1% Tween 20, at 4 °C overnight or room temperature for 1 h. Membranes were washed (× 3) in 1% TBS-Tween and incubated in HRP-conjugated swine anti-rabbit secondary antibody (P039901-2, 1:5000, Dako, Ely, UK), in 5% semi-skimmed milk/1% Tween 20, for 1 h at room temperature. Membranes were washed (× 3) in 1% TBS-Tween and TBS. Membranes were incubated in ECL™ Prime Detection Reagent (VWR International) and autoradiographic films (Hyperfilm™-ECL, Thermo Fisher Scientific) developed, per manufacturer’s instructions. Immunoblot images were captured and densitometry performed using ImageJ® Software, with untreated controls at each respective time-point representing 1.0-fold.

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