Western blot analysis of cell extracts and conditioned media of COS-7 cells transfected with fibrinogen cDNAs. (A) Samples of cell lysates and culture medium were subjected to 10% SDS-PAGE under reducing conditions (top panels) or 7.5% SDS-PAGE under nonreducing conditions (middle panels). The blots were incubated with polyclonal antihuman fibrinogen or monoclonal anti–β-actin antibodies (as a loading control for cell lysates in reducing conditions; bottom panel), and cross-reacting bands were revealed by chemiluminescence. Fg indicates purified fibrinogen control; -, COS-7 cells transfected with the empty vector; wt, COS-7 cells transfected with normal Aα, Bβ, and γ cDNAs; mut, COS-7 cells transfected with normal Aα, normal Bβ, plus mutant Arg134Xaa γ cDNAs. The positions of the hexameric complex and the normal Aα, Bβ, and γ chains are indicated. (B) Samples of cell lysates and culture medium were subjected to 12.5% SDS-PAGE under reducing conditions with shorter migration times. No truncated γ chain (predicted size, approximately 12 kD) is detectable.