Deregulated GSK3βactivation in CD34⁺ cells isolated from CML patients. (A) Primary CD34⁺ cells freshly isolated from healthy donors (CTR, n = 3) and CML patients in CP (n = 5) or BC (n = 3) were immunoblotted as indicated. Total levels of BCR-ABL and active β-catenin are also indicated. Total β-actin levels are reported as a loading control. (B) GSK3β was immunoprecipitated from CML progenitors (CD34⁺CD38⁺) and more primitive stem cells (CD34⁺CD38⁻) pooled from CML patients in CP (n = 5) or BC (n = 3) and incubated with (CP+ and BC+) or without (CP− and BC−) SB216763 5μM for 4 hours. Normal CD34⁺CD38⁻ cells were pooled from healthy donors (CTR, n = 3). GSK3β activity was examined by an in vitro kinase assay using histidine-tagged recombinant β-catenin (β-CateninHis) as a substrate of GSK3β. Phosphorylated β-catenin was detected by autoradiography (β-Catenin³²P). Levels of GSK3β and β-catenin were detected by Western blotting. (C) Representative histogram of intracellular pY²¹⁶ levels of GSK3β (GSK3β pY²¹⁶⁾ in CD34⁺ cells isolated from healthy donors (CTR; n = 3) and CML patients in CP (n = 5) or BC (n = 5). Background signal was assessed in the same populations by staining with a matched-isotype control. Mean fluorescence intensities (MFI) ± SD relative to GSK3β pY²¹⁶ signal in CD34⁺CD38⁺ progenitors and more primitive CD34⁺CD38⁻ cells from healthy donors (CTR; n = 3) and CML patients in CP (n = 5) or BC (n = 5) are shown. Significant differences in paired t tests for CD34⁺CD38⁺ cells versus CD34⁺CD38⁻ cells in CML BC patients are indicated (*P < .005).