Western blot analysis of cell extracts and conditioned media of COS-7 cells transfected with fibrinogen cDNAs.

Samples of cell lysates and culture medium were subjected to (A) 10% SDS-PAGE under reducing conditions or (B) 7.5% SDS-PAGE under nonreducing conditions. The blots were incubated with a polyclonal anti–human fibrinogen antibody and cross-reacting bands revealed by chemiluminescence, as described in “Study design.” Fbg indicates purified fibrinogen control; −, COS cells transfected with an empty vector. The positions of the hexameric complex and the normal Aα, Bβ, and γ chains are indicated; open arrowheads indicate probable α/γ intermediates (see “Results and discussion” and Hartwig and Danishefsky17). An asterisk marks the Trp467Stop mutant fibrinogen chain, which lacks 25 amino acid residues from the C-terminus. Transfections, lane 1: normal Aα, Bβ, and γ; lane 2: normal Aα, normal Bβ + mutant Trp467Stop Bβ, and normal γ; lane 3: normal Aα, mutant Trp467Stop Bβ (only), and normal γ.