Depletion of thiol-containing proteins inhibits TGF-β1 activation. (A) Thrombin-stimulated platelet releasates were incubated without and with shear of 1800 s⁻¹ for 2 hours and then labeled with MPB (100 μM) for 2 hours. MPB-labeled proteins in platelet releasates analyzed directly (non-IP) or after immunoprecipitation (IP) with an anti–TGF-β1. The MPB-labeled band at approximately 25 kD () was immunoprecipitated by anti–TGF-β1 and depleted in the supernatant after IP. (B) The 25-kD band corresponded to TGF-β1 detected by immunoblotting of the entire releasate (non-IP) and the anti–TGF-β1 immunoprecipitate (IP: TGF-β1). Vertical lines indicate deletion of intermediate lanes. (C) Platelet releasates were passed through either a control Sepharose column (con) or a thiol-Sepharose column (thiol) and then labeled with MPB and analyzed by SDS-PAGE, followed by Coomassie brilliant blue staining (left panel), blotting with streptavidin-HRP (to detect MPB; middle panel), or blotting with an anti–TGF-β1 antibody (right panel). Note that the majority of MPB-labeled proteins were depleted by passage over the thiol-Sepharose column and that TGF-β1 was partially depleted. A Coomassie brilliant blue–stained gel demonstrated that the thiol-Sepharose column did not deplete most of the platelet releasate proteins. (D) Platelet releasates that were passed through either a control Sepharose column (con) or a thiol-Sepharose column (thiol) were stirred and assayed for TGF-β1 activation by ELISA. Note that depletion of thiol-containing proteins reduced the percentage of TGF-β1 that could be activated by stirring. Error bars represent SD.