Transfections of 293 cells with fXI/pJVCMV expression constructs. FXI antigen levels in media (A) and cell lysates (B) of transient transfections are shown. 293 fibroblasts were transfected with 80 ng expression construct for individual transfections (columns 1-3) and 80 ng fXI-WT expression construct plus 80 ng construct to be tested for cotransfections (columns 4-6). The negative control in column 7 contains empty pJVCMV vector. Shown are means and standard deviations for 6 separate transfections, each measured in triplicate by ELISA and corrected for transfection efficiency by Renilla luciferase assay. The mean for single fXI-WT transfections was assigned a value of 100%. Lane (1) fXI-WT, (2) fXI-Phe225, (3) fXI-Tyr398, (4) fXI-WT + fXI-WT, (5) fXI-WT + fXI-Phe225, (6) fXI-WT + fXI-Tyr398, (7) pJVCMV without cDNA. Western blots for fXI in lysates of BHK cells transfected with pLg-fXI-IN constructs. Samples from stably transfected cell lines were run on a 7.5% polyacrylamide-SDS gel, followed by Western blotting with goat anti-human fXI polyclonal antibody. (C) Samples are (1) conditioned media from fXI-WT cells and lysates from cells transfected with (2) fXI-WT, (3) fXI-Phe225, (4) fXI-Glu350, and (5) untransfected BHK cells. (D) Samples are (1) conditioned media from fXI-WT cells and lysates from cells transfected with (2) fXI-WT, (3) fXI-Tyr398, (4) fXI-Ser569, and (5) untransfected BHK cells. Positions of dimeric (D) and monomeric (M) fXI are indicated to the right of each panel, and positions of molecular mass standards in kilodaltons are shown on the left. The ratio of dimer to monomer varied between experiments. Note that, with the exception of fXI-Glu350, which does not dimerize,⁵  patterns for mutants match the wild-type pattern.