Quantifying Intracellular Transglutaminase-2 Activity

TG2 activity was monitored by an in-situ assay as previously described [9 (link)]. For each cell culture, at least two independent experiments, each in triplicate, were performed. Briefly, fibroblasts were treated for 30 min at 37 °C with THP at concentrations 0.01, 0.1, and 0.5 µM, in the presence of the TG2 substrate pentylamine-biotin (Thermo Fisher Scientific, Monza, Italy) (0.5 mM); in some experiments, the TG2 inhibitor Z-DON (Zedira, Darmstadt, Germany) was employed at 40 µM. Then, cells were harvested in RIPA lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% SDS, 1 mM Na₃OV₄, 1 mM PMSF, and a cocktail of protease inhibitors). After lysis, proteins (25 µg) were coated overnight in wells of a 96-well microplate, incubated with a blocking solution (10% bovine albumin solution in borate buffer saline), then with horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific) 1:3000. Finally, peroxidase enzymatic reaction was performed by adding 3,3′,5,5′-tetramethylbenzidine. Absorbances measured at 450 nm were used as an estimation of the intracellular TG2 activity.