Analysis of SHP-2 basal activity and interaction with Src in normal and ET platelets. (A-B) Platelets from healthy donors (lanes 1-3) or ET patients (lanes 4-6) were disrupted by suspension in 1 mL of lysis buffer (lanes 1,4) or mild sonication in isotonic buffer and the cytosolic (lanes 2,5) and membrane fractions (lanes 3,6) were obtained as described in “Subcellular fractionation.” (A) The different fractions (15 μL) were analyzed by Western blot with anti–SHP-2, whose densitometric values are reported above the relative bands, or anti–SHP-1 or anti-Src antibodies. (B) The different fractions (400 μL) were immunoprecipitated with either anti-Src or anti–SHP-2 antibodies and the IPs were immunostained with anti–SHP-2 or anti-Src or anti-JAK2 antibodies. The panel is representative of data obtained from 12 normal and 12 ET samples (JAK2^V617F homozygous and heterozygous), except the immunostaining with JAK2 antibody, which was performed only in 4 normal and 4 ET samples. (C) Normal, PV, and ET platelets were incubated with vehicle (lanes 1-2), or 0.5mM pervanadate (lane 3) or 2 mg/mL calpeptin (lane 4) for 5 minutes. Cells were then lysed and the basal tyrosine phosphatase activity of the lysates was tested in vitro on [³³P]phospho band 3 of erythrocytes as detailed in “Tyrosine phosphatase assay.” Samples were then subjected to SDS/PAGE and the ³³P-phosphate bound to band 3 was evaluated by autoradiography followed by the counting of the excised ³³P-protein bands in a scintillation counter. The phosphatase activity is expressed as ³³P-phosphate released from ³³P-phospho band 3. Values represent the means of experiments performed in triplicate with 6 normal, 5 PV, and 7 ET samples. **P < .001 versus the phosphatase activity of normal platelets.