Induction of SHP-1 overexpression in KCL22-R cell line. (A) SHP-1 protein expression assessed by WB analysis on KCL22-S, KCL22-R^Control, and KCL22-R^SHP−1+ cell lines. Cell extracts are subjected to WB analysis with Ab-SHP-1 and Ab-ABL to detect the indicated proteins. Cell lines are treated with 5μM IMA for 24 hours. Lower panel shows densitometry analysis of the BCR-ABL–related signaling of 3 independent experiments. (B) Anti–SHP-2 IP of lysates from KCL22-S and KCL22-R^SHP−1+ cell lines, untreated or treated with 5μM IMA for 24 hours; proteins are separated on 10% SDS-PAGE. Membranes are immunoblotted by Ab-SHP-2 as control (top) and anti–SHP-1 (bottom). IP indicates immunoprecipitation by anti–SHP-2; C, immunoprecipitation by an irrelevant rabbit IgG. (C) Proliferation analysis on KCL22-R^Control and KCL22-R^SHP1+ cells treated with 5μM IMA for the indicated days of in vitro culture, with respect to untreated cells. Proliferation is assessed by MTS assay in triplicate wells, for 3 independent experiments. Results are expressed as mean ± SD. (D-E) WB analysis to evaluate phosphorylation status of SHP-2 Tyr542 in KCL22-S, KCL22-R, and KCL22-R^SHP−1+ cell lines. Cell lines are treated with 5μM IMA for 30 minutes, and protein lysates are separated on 10% SDS-PAGE. Membranes are immunoblotted with antibody against SHP-2 pTyr542 and SHP-2, as protein loading control. Figure shows 1 representative experiment (D), as well as densitometry analysis of 3 independent experiments (E). SHP-2 expression has been used to normalize the signal of the phosphorylated form. (F) SHP-2 activity is assessed by specific phosphatases assay in KCL22-S, -R^Control, R^SHP−1+ cell lines, 1 HD and 1 patient with CP-CML. Cell lines are treated with 5μM IMA for 24 hours. Results are expressed as mean ± SD of 2 independent experiments.