Pa5 95256

Protein samples were prepared by homogenizing snap frozen tissue in 1× RIPA (ThermoFisher) buffer containing protease inhibitors (Roche). Protein concentrations were determined using the Bradford assay (BioRad protein assay reagent). Aliquots of total protein (10 μg for aorta and liver; 50 μg for all other tissues) resolved by SDS–PAGE and electro-transferred to nitrocellulose membranes. Blots were blocked with 5% nonfat dry milk in TBST for 1 h at room temperature and then incubated overnight with primary antibody. After rinsing, the membranes were incubated with HRP-conjugated secondary antibodies (BioRad) for 1 h at room temperature followed by chemiluminescent detection (BioRad ChemiDoc). Antibodies used were TG2 polyclonal (PA5-95256; ThermoFisher) and GAPDH (1D4; NB300-221; Novus Biologicals). ImageLab (BioRad) was used for densitometry analysis.

TG2’s interaction with fibronectin and integrin β1 was determined by co-immunoprecipitation. Briefly, precleared liver homogenates (500 µg) were incubated with indicated antibody (0.5 µg) for 2 h at 4 °C with gentle rocking. The immunocomplexes were then enriched by incubating the samples with protein G-agarose beads (50 µL). The beads were rinsed three times to remove unbound proteins. Bound proteins were recovered by boiling the beads in 50 µL of 2× Laemmli buffer. Isotype antibody was used as a negative control. Immunoprecipitated proteins were resolved by SDS–PAGE, and immunocomplex-bound proteins were determined by western blotting. Antibodies used were fibronectin monoclonal (FBN11; Invitrogen), integrin β1 (CD29; HMβ1-1; BD Bioscience); and TG2 polyclonal (PA5-95256; ThermoFisher). Densitometry analysis was done using ImageLab software (BioRad).