Figure 1

Expression and characterization of transglutaminase 2 constructs in MCF10A cells. (A) Representative immunoblot of cell lysates showing transglutaminase 2 (TG2) expression in MCF10A sublines stably transfected with lentiviral vector alone (Vec) or lentiviral vector containing the wild-type (TG2-WT), transamidase-inactive (TG2-C277S) or GTP-binding inactive (TG2-R580A) TG2 construct. The membrane was stripped and reprobed with anti-actin antibody to ensure even protein loading. (B) Immunofluorescent staining for TG2 and 4',6-diamidino-2-phenylindole (blue) staining for the nuclei in the indicated MCF10A sublines. Original magnification ×200. (C) In situ transamidation activity of TG2 constructs was studied by preincubating the indicated MCF10A cells with 1 mM 5-(biotinamido)pentylamine (BPA) overnight, followed by 8 hours of incubation in medium alone (-) or medium containing 8 μM (+) calcium ionophore, A23187. Cells were harvested, and BPA-conjugated cellular proteins were detected by an immunoblotting type assay using horseradish peroxidase-conjugated streptavidin as a probe. The membrane was reprobed with anti-TG2 and β-actin antibody to ensure even loading and levels of TG2 expression. (D) Immunoblot showing GTP-binding ability of different TG2 constructs. Pull-down experiments were performed with GTP-agarose beads incubated for either 30 minutes (30') or overnight (o/n) to evaluate GTP-binding ability for the indicated TG2 forms. Input shows the level of TG2 expression in MCF10A cell lines expressing different TG2 constructs.