In vitro expression of wild-type and Leu172Gln mutant fibrinogens. Immunoprecipitated proteins from conditioned media (A) and cell lysates (B) of COS-1 cells transfected with equimolar mixtures of plasmids driving the expression of the wild-type Aα and γ chains, together with the wild-type or Leu172Gln Bβ chains. Untransfected HepG2 cells and mock-transfected (with the unrelated pUC18 plasmid) COS-1 cells represent the positive and negative controls, respectively. Cells were labeled with [³⁵S]-methionine and [³⁵S]-cysteine for 2 hours and subsequently chased for 0, 30, 60, 120, and 240 minutes. Samples were separated on 4% SDS-PAGE under nonreducing conditions. Labeled proteins were visualized by exposing gels overnight to a storage phosphor screen and analyzed using a Typhoon 9200 phosphor imager and the ImageQuant software (Amersham Pharmacia Biotech, Uppsala, Sweden). The arrowheads indicate the 340-kDa hexameric fibrinogen molecule. A densitometric analysis of the bands corresponding to wild-type (□) and mutant (▦) immunoprecipitated fibrinogen is shown on the right. Bar graphs are expressed as arbitrary densitometry units (y-axis), as calculated by the ImageQuant software by integrating intensities of all the pixels in the band excluding the background. To compare samples from different panels, each value was normalized using the intensity of the HepG2 fibrinogen band from the same panel. (C) Measurement of fibrinogen antigen levels in conditioned media and in cell lysates by means of an ELISA assay, 64 hours after transfection. Bars represent means ± SD of 3 independent experiments. Fibrinogen levels are reported as ng/mL (Y-axis).