Transfection studies. (A) 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 10% SDS-PAGE under reducing conditions or 7.5% SDS-PAGE under nonreducing conditions. The blots were incubated with a polyclonal antihuman fibrinogen antibody and cross-reacting bands were revealed by chemiluminescence, as described.¹⁰  Fib 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. Lane 1: normal Aα, Bβ, and γ; lane 2: normal Aα, normal Bβ plus mutant G444S Bβ, and normal γ; lane 3: normal Aα, mutant G444S Bβ (only), and normal γ. (B) Multiple alignment of the C-terminal region of fibrinogen Bβ chain. Sequences from human (P02675), mouse (XP_130960), rat (P14480), bovine (P02676), chicken (Q02020), Xenopus (Q91589), and lamprey (P02678) were obtained from the Swiss-Prot database (except the mouse sequence, which was obtained from the National Center for Biotechnology Information [NCBI]) and aligned using the ClustalW program (http://www.ebi.ac.uk/clustalw/). Conserved amino acids are highlighted in gray; arrows indicate the positions of previously reported mutations: L383R, G430D, Y447G, W467X, W470X. The open arrow indicates the position of the mutated glycine residue (G444S).