Expression and activation of LYN and HCK kinases in STI571-resistant CML patients.

(A) Cell lysates were prepared from K562-R cells and specimens derived from several patients who failed to sustain hematologic remission with STI571 daily therapy (400-600 mg). Equal protein aliquots (20 μg) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for LYN or HCK with specific primary antibodies as described in “Materials and methods.” Actin levels on the same blot were used to determine protein equivalence in each sample. (B) Blood samples were collected from blast-crisis CML patients one day prior to beginning STI571 daily therapy (day 0) and at 1 or 2 points after disease progression (as noted). Specimens were prepared (as described in “Materials and methods”), protein content was estimated, and equal protein extracts were resolved by SDS-PAGE and immunoblotted with antibodies recognizing phosphorylated LYN (or HCK; p-HCK, p-LYN). The blot was stripped of primary antibody and reprobed with anti-LYN. Actin also was probed to determine protein equivalence in each sample.