Bcr-Abl activity detection using Abl substrate peptide in the human CML cell line K562.

<p>Bcr-Abl activity was detected using the biosensor peptide in K562 cells at 5, 30 and 60 min (N = 3). Peptide and phosphopeptide were detected below the 15 kD molecular weight marker by overlaid signal from IR-dye-labeled streptavidin (showing biotinylated peptide) and Western blot for antiphosphotyrosine (4G10). Phosphorylated endogenous proteins Abl, STAT5 and CrkL, as well as β-tubulin as a loading control, were detected as described in the Materials and Methods. Additional replicate blots shown in the supporting information. Quantification of total peptide (streptavidin bands) and phosphopeptide (4G10 bands) via raw data from integration of fluorescence intensity at the MW observed for the biosensor (without background subtraction) is shown (B) to illustrate degradation of the peptide in the presence of phosphatase inhibitor, which may have contributed to the decrease in phosphopeptide signal over time (C). White = peptide alone, grey = inhibition by Gleevec (imatinib/IM), black = phosphatase inhibitor sodium pervanadate (PV). Error bars represent SEM from three independent experiments.