Figure 3.
Metabolic radiolabeling and immunoprecipitation of platelet PAI-1. (A) Isolated platelets were incubated in the presence of 35S-methionine for 1, 3, and 6 hours. Platelet lysate and medium were immunoprecipitated with PAI-1 (ab-1) and the precipitated protein was separated with SDS-PAGE and subsequently examined by autoradiography. This yielded a protein of the expected molecular mass (approximately 45 kD), and the increasing amount of radioactive PAI-1 over time confirmed that there is an ongoing synthesis. (B) To inhibit protein synthesis, platelets were incubated for 6 hours in the presence of 1 mM puromycin, resulting in attenuated PAI-1 expression.
