Fig. 1. Cell-surface tTG promotes Fn assembly independent of its crosslinking activity. Populations of Swiss 3T3 transfectants (vector, tTG[1], tTG[2], tTG_C277S[1] and tTG_C277S[2]) were analyzed for integrin-tTG association and Fn biosynthesis and assembly. (A) Association of transfected tTG or its catalytically inactive mutant tTG(C277S) with endogenous 5ß1 integrin. 5ß1 integrin and tTG were immunoprecipitated from cell lysates with mAb BMA5 and polyclonal anti-tTG antibody, respectively. The resulting immunoprecipitates were blotted for tTG. (B) Biosynthesis of Fn in the transfectants. Fn was immunoprecipitated from ³⁵S-labeled cell lysates and analyzed by SDS-PAGE and autoradiography. (C,D) tTG and tTG(C277S) stimulate incorporation of [¹²⁵I]Fn into the deoxycholate-insoluble fraction. (C) Deoxycholate-insoluble [¹²⁵I]Fn in the matrix of cells grown for 24 hours with 10 nM [¹²⁵I]Fn was analyzed by reducing SDS-PAGE and autoradiography. Arrow points to Fn monomer; arrowhead marks tTG-crosslinked Fn polymers. Vector, tTG[1], tTG[2], tTG_C277S[1] and tTG_C277S[2] transfectants are marked as 1, 2, 3, 4 and 5, respectively (B,C). (D) ¹²⁵I-labeled bands corresponding to Fn monomer and polymer were cut out of the gels and counted. The results are representative of three independent experiments (mean±s.e.m.). (E) tTG and tTG(C277S) promote Fn fibrillogenesis. Cells grown for 24 hours in the presence of 50 nM exogenous Fn were stained with anti-Fn antibody. Bar, 50 µm.