SDS-polyacrylamide gel electrophoresis, western immunoblots, and ELISA of human factor XI. (A left panel) GelCode blue–stained 10% polyacrylamide–sodium dodecyl sulfate gel of plasma (pXI) or recombinant (rXI) human fXI (2-4 μg) purified using a monoclonal IgG against the A3 domain (pXI) or protease domain (rXI). (A right panel) Recombinant fXI (4 μg) run unreduced (U) or reduced (R). MW indicates molecular weight markers. (B) Ribbon model of a hypothetical hybrid apple domain encoded by the fXI Δ6/7 mRNA, based on the primary amino acid sequence encoded by exons 5 (black ribbon) and 8 (white ribbon) and the crystal structure of human fXI.⁵  The domain is bounded by a disulfide bond between Cys92 and Cys265. Strands 2-7 of the β-sheet are numbered. (C) Chemiluminescent Western blots of plasma (pXI) or recombinant (rXI) fXI using goat polyclonal anti–factor XI IgG (left panel), monoclonal IgG O1A6 (recognizes A3 domain; center panel), or monoclonal IgG 14E11 (recognizes A2 domain; right panel) as the primary antibody. (D) ELISA of cell lysates from HEK293 fibroblasts transiently transfected with expression constructs for WT-fXI (□), fXI-Δ6/7 (), or empty vector (pJVCMV^6,7 ; ■). The ELISA capture antibodies were a goat polyclonal anti–human fXI IgG (Poly), O1A6, 14E11, or a monoclonal antibody against the protease domain (1G5). The detection antibody was a goat polyclonal anti–human fXI IgG conjugated to horseradish peroxidase. (E) Chemiluminescent Western blots of immunoprecipitates of lysates of HEK293 cells transfected with WT-fXI (WT), fXI-Δ6/7 (Δ6/7), or empty vector (C). pXI is a plasma fXI control. Immunoprecipitation was done with IgG 1G5.^6,7  Primary antibodies are indicated below each blot. For panels A, C, and E, positions of molecular mass standards (in kDa) are indicated on the left. D indicates fXI dimer; M, fXI monomer; X, putative fXI Δ6/7 splice variant; and 6/7, fXI-6/7 splice variant.