Pentylamine biotin

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.

Oligonucleotides were synthesized either by Greiner Bio-One, Integrated DNA Technologies or by Eurofins. Restriction enzymes, DNA polymerases, Klenow fragment and Recombinant RNase Inhibitor were purchased from Takara Bio. Recombinant Transglutaminase 2 was from Novus Biologicals, USA. Components of in vitro cell-free protein synthesis kit, PUREfrex, were provided by GeneFrontier, Japan. Puromycin cnvK linker was obtained from Epsilon Molecular Engineering Inc., Japan. Pentylamine-biotin, RNase T1, SYBRGold, and streptavidin-coated magnetic beads streptavidin MyOne C1 were purchased from Thermo Fisher Scientific. N-terminal biotinylated peptides Top 1, Top 2, T26, T26QN and peptide with heatmap-derived sequence were chemically synthesized by Bio-Synthesis, USA and provided by Biologica, Japan.

In situ TG2 activity was detected by a microplate assay using the TG2 substrate pentylamine-biotin (Thermo Fisher Scientific) as reported elsewhere [37 (link)], with some modifications. Briefly, fibroblasts were seeded at a density of 3000/cm² in plates of 60 mm in diameter and cultured for 48 h, then treated with different amounts of p31–43, p57–68, and the control peptide p229–246 for 30 min. At the end of the incubation with peptides, cells were harvested in RIPA lysis buffer, consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 0,1% SDS, 1 mM Na₃OV₄, 1 mM PMSF, and an inhibitor cocktail of proteases. Total lysates were centrifuged at 12,000× g for 10 min at 4 °C. Supernatants (25 μg) were used to coat wells of a 96 well microplate. A blocking solution consisting of 10% bovine albumin in borate buffer saline (80 mM NaCl, 100 mM H₃BO_3, 20 mM Na₂B₄O₇) was added to each well for 2 h. After several washes, wells were treated with horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific) 1:3000 in borate buffer saline with 0.1% Tween 20, then color was developed by adding 3,3′,5,5′-tetramethylbenzidine. By measuring absorbances at 450 nm, we obtained an estimation of intracellular TG2 activity, in the absence and in the presence of gliadin peptides.