Western blot analysis of PASMC-secreted TFPI. (A) Quiescent PASMC were treated with basal medium alone (lanes 3 through 5) or the basal medium containing bFGF/heparin (10 ng/15 U/mL; lanes 6 through 8) or serum (10% vol/vol; lanes 10 through 12) for 8 (lanes 3, 6, and 10), 24 (lanes 4, 7, and 11), and 48 hours (lanes 5, 7, and 12). Other lanes are as follows: lane 1, full-length recombinant TFPI from BHK cells; lane 2, molecular weight markers; and lane 9 (zero time), aliquot from the serum-containing medium that was removed immediately after its addition to the cells. The blot was probed with monospecific polyclonal anti-TFPI IgG. (B) One hundred microliters of conditioned media from cells treated for 48 hours with serum-free (control), bFGF (10 ng/mL)/heparin (15 U/mL) (bFGF), and serum (10% vol/vol)-containing media were subjected to SDS-PAGE followed by Western blot analysis. Other lanes are as follows: full-length recombinant TFPI from BHK cells, diluted either in a buffer (rTFPI) or in control serum (10% vol/vol). The blot was probed with a 100-fold diluted rabbit antiserum raised against TFPI C-terminal domain peptide. Molecular weight markers are Bio-Rad prestained markers (low range).