Figure 2. BCR-ABL signaling activity is not fully restored to baseline levels in treatment/washout conditions of dasatinib and ponatinib that commit cells to apoptosis.

K562 cells were incubated alone or in the presence of 10 and 100 nM dasatinib or ponatinib for 2 h, subjected to standard and expanded washout, and collected at the indicated timepoint post washout for analysis by (A) immunoblot and (B) Phosflow FACS analyses. For immunoblot analyses, the phosphorylated and non-phosphorylated forms of CrkL were resolved by SDS-PAGE, blotted using a total CrkL antibody, and results are expressed as % pCrkL with the red, dashed line indicating the level of % pCrkL in untreated K562 cells. For Phosflow FACS analyses, cells were fixed, permeabilized, and stained using Alexa647-pCrkL and Alexa488-pSTAT5 conjugated antibodies. Results are displayed, for comparison purposes, as the overlaid signal peak traces of isotype control, untreated cells, and each indicated timepoint post washout. Vertical, black dashed lines highlight the peak signal in untreated K562 cells for reference.