![Figure 5. Inhibition of calpain activity rescues E coli–induced Bcl-xL degradation in platelets. (A-B) Platelets were left alone or pretreated with specific inhibitors (10μM epoxomicin [EPO]; 25μM lactacystin [Lact]; 10μM MG132; 50μM calpeptin [Calp]; 50μM E64d; and 50μM MDL28170 [MDL]) before being stimulated with A23187 (1μM) for 1 hour and intracellular Bcl-xL protein was assessed by Western analysis (A) or quantified by ELISA (B). The bars for panel B show the mean ± SEM of 4 independent experiments, and the asterisk indicates a significant decrease (P < .01) in Bcl-xL protein levels compared with control. (C-D) Platelets were pretreated with inhibitors (A and B) and then left alone (control) or incubated with wild-type UTI89 for 4 hours. Intracellular Bcl-xL protein was subsequently assessed by Western analysis (C) or quantified by ELISA (D). The bars for panel D depict the mean ± SEM of 4 independent experiments, and the single asterisk indicates a significant decrease (P < .01) in Bcl-xL protein levels compared with control. (E) Platelets were incubated in the presence or absence of EGTA (5mM) for 15 minutes and then left alone or stimulated with 1μM A23187, UTI89, or UTI89 ΔhlyA for 2 hours and Bcl-xL protein was assessed by Western analysis. This blot is representative of 3 independent experiments. (F) Whole blood was incubated with UTI89 in the presence or absence of calpeptin. After 4 hours, platelets were isolated, and intracellular Bcl-xL protein was measured by ELISA. The bars represent the mean ± SEM of 3 independent experiments, and the single asterisk indicates a significant decrease (P < .05) in Bcl-xL protein levels compared with control.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/25/10.1182_blood-2012-04-420661/4/zh89991200120005.jpeg?Expires=1722784962&Signature=szugoETf4~EJnIdiXBuueKnJxV3ntfzjU1smDWxT6W9PBPaB-IxqkTPMYao1iw2lgZWiwWr9-kPmDjDI7b5WjSWenfmtA7MJDWa7RzTvNT6TOiBhxc~~khifLl8TH79Ij3E2WW7kHXbuNS3lF1gGzFOf3BLfFldKbq8AH1ZtR6Ppol2kw7dI6X9rmf9b0AcF4zbikZjk7Q5m0I-PAB3UMdHUX3h5cF6l9B3WxRhcGCzNsd09mpxzRdYU0y5Yb2to~hKLmxlLM~xd9VcWQsS9K245k588ftCwVAjA6iT7hSA-EJjWfZVHbwj2RrZRBVKoh5HaI5hWq98zqJrnveGrLA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Inhibition of calpain activity rescues E coli–induced Bcl-xL degradation in platelets. (A-B) Platelets were left alone or pretreated with specific inhibitors (10μM epoxomicin [EPO]; 25μM lactacystin [Lact]; 10μM MG132; 50μM calpeptin [Calp]; 50μM E64d; and 50μM MDL28170 [MDL]) before being stimulated with A23187 (1μM) for 1 hour and intracellular Bcl-xL protein was assessed by Western analysis (A) or quantified by ELISA (B). The bars for panel B show the mean ± SEM of 4 independent experiments, and the asterisk indicates a significant decrease (P < .01) in Bcl-xL protein levels compared with control. (C-D) Platelets were pretreated with inhibitors (A and B) and then left alone (control) or incubated with wild-type UTI89 for 4 hours. Intracellular Bcl-xL protein was subsequently assessed by Western analysis (C) or quantified by ELISA (D). The bars for panel D depict the mean ± SEM of 4 independent experiments, and the single asterisk indicates a significant decrease (P < .01) in Bcl-xL protein levels compared with control. (E) Platelets were incubated in the presence or absence of EGTA (5mM) for 15 minutes and then left alone or stimulated with 1μM A23187, UTI89, or UTI89 ΔhlyA for 2 hours and Bcl-xL protein was assessed by Western analysis. This blot is representative of 3 independent experiments. (F) Whole blood was incubated with UTI89 in the presence or absence of calpeptin. After 4 hours, platelets were isolated, and intracellular Bcl-xL protein was measured by ELISA. The bars represent the mean ± SEM of 3 independent experiments, and the single asterisk indicates a significant decrease (P < .05) in Bcl-xL protein levels compared with control.
