Alliance 2.7 system

Placenta samples were homogenised in protein extraction buffer (100 mM Tris Base, 10 mM Na₄P₂O₇, 100 mM FNa, 1 mM Na₃ VO₄, 10 mM EDTA, 2 mM PMSF, 0.1 mg/mL aprotinin, 1 % Triton X-100, pH 7.4) followed by centrifugation at 10,000 × g for 15 min at 4 °C. Placental proteins (40 μg) were resolved by 12 % SDS-PAGE at 90 V for 1 h, followed by transfer onto 0.45-μm nitrocellulose membranes at 300 mA for 2 h. The membranes were blocked with 5 % non-fat dry milk in Tris-buffered saline (pH 7.5) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies against the 20S, 19S and 11S proteasome subunits (#PW8165, #PW8195, Affinity, USA; 1:1500 dilution) as well as MuRF-1 (#SC-32920) and MAFbx (#SC-33782; both from Santa Cruz Biotechnology, Heidelberg, Germany; 1:200 dilution). Immunoreactivity was detected by sequentially incubating the membranes with specific secondary antibodies (1:10,000 dilution, Cell Signaling Technology) for 1 h at room temperature and visualised using a chemiluminescence detection system. The blots were scanned using a gel image capture system to quantify differences via densitometry (Alliance 2.7 system, Alliance 1D capture software, and UVIBand 12.14 analysis software; UVITEC, Cambridge, UK). The levels of all detected proteins were reported relative to the level of 34-kDa GAPDH.

Proteins from untreated and MPs-treated hGFs (for 48 h) 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-NFkB (1:500; Santa Cruz Biotechnology), mouse anti-MyD88 (1:500; Santa Cruz Biotechnology) and mouse anti-NLRP3 (3 µg/mL; Novus). Subsequently, membranes were incubated for 60 min at room temperature with peroxidase-conjugated anti-mouse secondary antibody (1:5000; Bethyl Laboratories, Montgomery, AL, USA) [35 (link)]. Enhanced chemiluminescence with the Alliance 2.7 system (Uvitec Ltd., Cambridge, UK) was used to identify and quantify the bands obtained.

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.