Figure 4.

Bengamide A Reduces c-Src Tyrosine Kinase Activity.

All cells were treated with DMSO, IV-43 (10 μM), TNP-470 (100 nM), bengamide A (10 nM) and the combination of IV-43 (10 μM) and TNP-470 (100 nM) for 24 hours before harvest. (A). Acid-denatured enolase was used in the in vitro kinase assay for immunoprecipitated extrageneous c-Src autoradiography (upper panel), Commassie blue-stained enolase (middle panel) and anti-myc immunoblot (lower panel) are shown as gel-loading controls. (B) and (C). Immunoprecipitated endogeneous c-Src autoradiography is shown in the upper panel. Commassie blue-stained enolase (middle panel) and anti-Src immunoblot (lower panel) are shown as gel-loading controls. +Bengamide A represents the addition of bengamide A (100 nM) into the immunoprecipitates before the initiation of the kinase assay. (D) HeLa cell lysate was immunoblotted by anti-phospho-Tyr419 and anti-Tyr530. Immunoblots of total c-Src and β-actin are shown as gel loading controls. Na₃PO₄ (2 mM) was added to the culture medium for 15 min before harvesting the cells. (E) Immunoblotting of HeLa total cell lysate by anti-phosphotyrosine (clone 4G10). Arrows indicated the positions of change with 24-hours bengamide A (100 nM) treatment. Tubulin immunoblot is shown as the gel loading control. (F). Src++ MEF cell lysate is immunoblotted by anti-Tyr530 as an indication of active c-Src in Src++ MEF. Bengamide A (10 nM) was added to the culture medium for 24 hours. Na₃PO₄ (2 mM) was added to the medium for 15 min before harvesting the cells. Immunoblot of total c-Src is shown as gel loading control.