Profiling Inflammatory Mediators in hGFs

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.