Proteins from untreated and LPS-G- and/or AA-treated hGMSCs and e-hGMSCs were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot analysis (Bio-Rad V3 Western Workflow™, Milan, Italy).
Membranes were saturated for 120 min at room temperature in a blocking buffer (1× TBS, 5% milk, 0.1% Tween-20) followed by overnight incubation at 4 °C with the following primary antibodies: mouse anti-p300 (1:750; OriGene) and mouse anti-DNMT1 (1:750; OriGene). Subsequently, membranes were incubated for 60 min at room temperature with peroxidase-conjugated anti-mouse secondary antibody (1:5000; Bethyl Laboratories, Montgomery, AL, USA) (Diomede et al. 2016 (link)). Enhanced chemiluminescence with the Alliance 2.7 system (Uvitec Ltd, Cambridge, UK) was used to identify and quantify the bands obtained.
Membranes were saturated for 120 min at room temperature in a blocking buffer (1× TBS, 5% milk, 0.1% Tween-20) followed by overnight incubation at 4 °C with the following primary antibodies: mouse anti-p300 (1:750; OriGene) and mouse anti-DNMT1 (1:750; OriGene). Subsequently, membranes were incubated for 60 min at room temperature with peroxidase-conjugated anti-mouse secondary antibody (1:5000; Bethyl Laboratories, Montgomery, AL, USA) (Diomede et al. 2016 (link)). Enhanced chemiluminescence with the Alliance 2.7 system (Uvitec Ltd, Cambridge, UK) was used to identify and quantify the bands obtained.