Effect of STI-571 on bcr-abl phosphorylation, Stat-5 activation, bcl-xL, and PTP1B expression in Mo7e and MBA cells; comparison with other kinase inhibitors in bcr-abl+K562 cells.
(A) Mo7e or MBA cells were cultured in the presence of GM-CSF (200 U/mL) and treated with 0, 1, or 10 μM STI-571 for 60 minutes before equal-protein (20 μg) cell lysates were analyzed for Stat-5 and bcr-abl tyrosine phosphorylation by immunoblotting (anti-phospho–Stat-5 recognizes tyrosine-phosphorylated bcr-abl). After phosphoprotein detection, the blot was stripped and reprobed with anti–Stat-5 (bottom). (B) MBA cells were incubated with 1 μM STI-571 for 0, 2, or 4 hours before cells were harvested and nuclear proteins extracted (see “Materials and methods”). Consensus Stat-5 DNA binding oligomers (w, wild-type; m, mutant sequence; Santa Cruz Biotechnology) were radiolabeled to 5 × 106 cpm/pmol DNA and incubated with 5 μg nuclear protein from control or STI-571–treated cells. DNA–protein complexes were resolved by EMSA on native polyacrylamide gels (6.5%). Arrows depict Stat-5–complex and nonspecific binding complexes (NSB). Radioactivity in the Stat-5–DNA complex was quantitated by PhosphorImager. (C) Mo7e or MBA cells were treated with 1 μM STI-571 for the interval indicated before cell lysates were harvested and immunoblotted for bcl-xL, PTP1B, or actin as a protein-loading control (top). K562 cells were incubated with the indicated concentration of STI-571 for 16 hours before equal-protein cell lysates were analyzed for bcl-xL levels or β-actin as a protein loading control (bottom). (D) K562 cells were treated as indicated for 60 minutes before cell lysates were analyzed for altered signaling processes by immunoblotting. Equal-protein lysates (20 μg) were sequentially blotted with phospho–Stat-5, phospho-MAPK, and phospho-Akt or β-actin as a protein-loading control.
