SHP-1 expression in KCL22 CML cell lines and its interaction with SHP-2. (A) Quantitative evaluation by RT-qPCR of BCR-ABL, SHP-1, SHP-2, PP2Ac, and SET mRNA expression in KCL22-R and -S cell lines. The experiments are performed in triplicate, and results are indicated as mean ± SD. (B) WB analysis on total protein lysates of KCL22-R and KCL22-S cell lines. Proteins are separated on 10% SDS-PAGE and immunoblotted with antibodies against ABL, pABL (pTyr245), SHP-1, SHP-2, pCrkL (pTyr207), PP2Ac, and SET proteins. GAPDH is used as protein loading control. Cell lines are treated with 5μM IMA for 24 hours, with the exception of the cell lysates analyzed for phospho-protein, for which cell lines are treated with 5μM IMA for 30 minutes. (C-E) Anti–SHP-2 immunoprecipatation in KCL22-S and KCL22-R cell lines. Cellular lysates from KCL22-S and KCL22-R cell lines, untreated or treated with 5μM IMA for 24 hours, are immunoprecipitated by Ab-SHP-2 and separated on 10% SDS-PAGE. Membranes are immunoblotted by Ab-SHP-2 as control (C) and by Ab-SHP-1 (D). Total cell lysates are analyzed for the presence of SHP-1 in the input loading by immunoblot with Ab-SHP-1 (E). GAPDH is used as protein loading control. (F-G) KCL22 cell lines are treated with 5μM IMA for 30 minutes, and protein lysates were separated on 10% SDS-PAGE. Membranes are immunoblotted first with Ab-SHP-2 pTyr542, and then with Ab-SHP-2, as protein loading control. Figure shows 1 representative experiment (F), as well as densitometry analysis of 4 independent experiments (G). Input indicates cell lysates without immunoprecipitation; IP, immunoprecipitation by anti–SHP-2; and C, immunoprecipitation by an irrelevant rabbit IgG.