c-Abl is phosphorylated and undergoes subcellular localization change in the presence of LatB and RGDfV. (A-D) HBMECs (2 × 105 cells/well) were seeded on VN-coated/HD-BSA–blocked 6-well plates in 0.4% BSA/RPMI 1640 and treated with LatB (0.5μM; A,C) or RGDfV (5 μg/mL; B,D) for the times indicated. (A,C) The short time points (5-30 minutes) are labeled as 5′, 10′, and 30′, and the remainder of the lanes were incubated for 2 to 24 hours. Where indicated, STI-571 (5μM) was also included in the medium, starting 30 minutes before LatB or RGDfV. Whole-cell lysates were resolved on 10% SDS-PAGE and analyzed by Western blotting for c-Abl (SH2 domain, 8E9 antibody) and tyrosine phosphorylation, with either phospho-c-Abl-Y412 (A-B) or phospho-c-Abl-Y245 (C-D). GAPDH served as loading control. (E) To examine c-Abl translocation, HBMECs (3000 cells/well) were allowed to adhere and spread overnight on VN-coated/HD-BSA–blocked 8-well chamber slides. LatB (0.2μM) was added in 0.4% BSA/RPMI 1640 for 0.5, 4, or 24 hours. Cells were permeabilized and stained with phalloidin (actin, red), c-Abl (8E9, green), counterstained with DAPI (nucleus, blue), and photographed. Shown is representative field (original magnification ×400).
