Enhancement of p42/44 MAPK phosphorylation in CML CD34⁺ cells by imatinib treatment. (A) CML and normal CD34⁺ cells (2 × 10⁵) were exposed to imatinib (1 and 5 μM) for 16 hours in medium containing low concentrations of GFs. Whole-cell lysates were prepared, electrophoresed on 10% SDS-PAGE, and Western blotted with antibodies to dual phosphorylated p42/44 MAPK (P-MAPK). Blots were stripped and reprobed with antibodies to total p42/44 MAPK (MAPK). Representative results for 2 CML samples and a normal control sample are shown. (B) Cumulative results for 6 CML and 3 normal samples are shown. P-MAPK/MAPK ratios were calculated by densitometry. Mean ± SEM of P-MAPK/MAPK ratio for treated cells expressed relative to controls is shown for CML (▪) and normal (▦) samples. Significant differences in a paired t test for treated CD34⁺ cells compared with untreated controls are indicated (^*P < .07; ^**P < .02). (C) MAPK kinase assays were performed on CML CD34⁺ cells cultured with low concentrations of GFs in the absence and presence of imatinib. The p42/44 MAPK was immunoprecipitated, and in vitro kinase assays were performed with the use of an ELK-1 fusion protein as substrate followed by Western blot with an anti–phospho-ELK-1 antibody. Representative results for 2 CML samples are shown. (D) Results of Western blotting for P-MAPK and MAPK in CML and normal CD34⁺ cells cultured with or without imatinib (5 μM) in medium without addition of GFs. (E) Results of Western blotting for P-MAPK and MAPK in CML and normal CD34⁺ cells cultured with or without imatinib (5 μM) in medium containing high (25-fold higher) compared with low concentrations of GFs.