Evaluation of SHP-1 expression at protein level in patients with CML. (A) SHP-1 expression is assessed by WB analysis in patients with CML. Protein lysates of BM aspirates derived from 4 patients classified as failure responders and 4 optimal responders are separated on 10% SDS-PAGE and immunoblotted with Ab-SHP-1. GAPDH is used as protein loading control. (B) Densitometric analysis of the SHP-1 immunoblot shown in panel A. Failure responder patients express a significant lower amount of SHP-1 protein expression compared with optimal responders (P = .007). (C) SHP-1 mRNA expression level assessed by RT-qPCR in the same patients with CML analyzed in panels A-B. Failure responder patients express a significant lower amount of SHP-1 mRNA compared with optimal responders (P = .003). (D) MSP analysis done on SHP-1 promoter region of patients with CML (1-6 = optimal responders; 7-12 = failure responders). Each sample is amplified by methylated primers (M) or unmethylated primers (U). NTC indicates nontemplate control; MC, methylated control; UC, unmethylated control; MW, marker of molecular weight. (E) Anti–SHP-2 IP analysis in CD34⁺ cells derived from 2 optimal responder and 2 failure responder patients. Protein lysates were normalized by GAPDH staining before IP experiments. (Top) Control immunoblot of IP lysates by staining with Ab–SHP-2; (middle) anti–SHP-1 immunoblot; total lysates are analyzed for the presence of SHP-1 protein by immunoblot with Ab–SHP-1 (bottom).