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Blood, Vol. 111, Issue 5, 2667-2673, March 1, 2008
Activated protein C–cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin
Blood Schuepbach et al.
111: 2667
Supplemental materials for: Schuepbach et al
Files in this Data Supplement:
- Figure S1. Immunoassays specifically detect surface PAR1 but not intracellular pools (JPG, 45.9 KB)
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(A) Confluent EAhy.926 cells were incubated for 1 h either without or with 1 nM thrombin, fixed and stained with WEDE15 using identical conditions as for the ELISA. WEDE15 binding was visualized by an AlexaFluor488 labeled goat anti mouse antibody. Incubation with thrombin abolishes extracellular staining (control) and the WEDE15-blocking peptide prevents form specific staining. Representative images are shown (3 independent repeats). Similar data was obtained using anti-PAR1 ATAP2 (not shown). (B) Confluent EAhy.926 cells were incubated as described above followed by cell surface biotinylation. Cell extracts were either analyzed directly (cell extracts) or after 30 minutes of incubation with streptavidin agarose (supernatants). Proteins pulled down by streptavidin agarose were eluted in reducing sample buffer at half the i.p volume. Anti PAR1 (ATAP2) immunoreactive bands are shown as well as anti- -actin immunoreactive bands of the same reprobed membrane. -actin staining is unaffected by agonist incubation, biotinylation and streptavidin precipitation and streptavidin agarose does not pull down this intracellular protein. Cell surface biotinylation does not affect PAR1 immunoreactive bands in total lysates, decreases the amount of PAR1 detected in the supernatants after streptavidin ip. and mediates PAR1 binding to streptavidin agarose. Upon incubation with thrombin a smaller PAR1 immunoreactive product is generated. The results were confirmed in 3 independent experiments.
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