Fig. 1

A 50 kDa protein, identified as uPA, associates with the extracellular domain of TEM8. a Human hepatoma tissue lysates were applied to a TEM8-Fc -conjugated Protein A Sepharose affinity column. Purified TEM8-Fc and the eluate were separated by 12% SDS-PAGE and stained with Coomassie brilliant blue. The dotted arrow indicates a TEM8-Fc -binding protein of approximately 50 kDa. b Sequence of the uPA protein. The amino acid residues of tryptic peptides identified by mass spectrometry are underlined or enclosed in boxes. c The hepatoma tissue homogenate was immunoprecipitated with TEM8-Fc or human IgG as a control, and binding to uPA was detected by immunoblotting with an anti-uPA antibody. d The hepatoma tissue homogenate was immunoprecipitated with anti-TEM8 or anti-uPA antibodies, or isotypic IgG as a control, followed by immunoblotting with an antibody recognizing TEM8. e The HepG2 cells were treated with uPA for 2 h followed by ultrasonication to harvest the cell lysate. The lysate was immmunoprecipitated by TEM8 and then detected by immunoblotting using TEM8 or uPA antibody. f The extent of binding of increasing concentrations of TEM8-Fc to the wells of microtiter plates coated with 0.1 μg of purified PA, HMW-scuPA, HMW-tcuPA or LMW-uPA in the presence of 2 mM MnCl₂. The data are expressed as means of triplicate samples analyzed from three representative experiments