Figure 3.

Expression of exogenous tTG or its enzymatically inactive mutant tTG[C₂₇₇→S], but not of FXIIIa promotes cell spreading. (A–E) REF52 cells were transfected with vector (vect.), wild-type tTG (tTG), tTG noncatalytic mutant C₂₇₇→S (tTG[C₂₇₇-S]), or FXIIIa (FXIIIa). (A) Expression levels of tTG on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Expression levels of FXIIIa on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal antibody against FXIIIa and flow cytometry. (C) Transglutaminase activity in the cytosolic fractions of the transfectants was quantified by incorporation of [³H]putrescine into N,N-dimethylcaseine by transamidating enzymes present in cell lysates (Lorand et al. 1972). Shown are the results of triplicate determinations. (D) Transglutaminase activity on the surface of live transfectants was quantified by measuring cell-mediated incorporation of [³H]putrescine into N,N-dimethylcaseine. Bars represent the means of triplicate measurements. (E) Cells expressing vector, tTG, tTG[C₂₇₇→S], or FXIIIa were photographed in regular culture. Bar, 20 μM.