Figure 3
Figure 3. Analysis of Src activation state in resting and thrombin-stimulated platelets. (A) Platelets, isolated from healthy donors or PV and ET patients, were lysed in IP-buffer and immunoprecipitated with anti-Src, anti-Fyn, or anti-Lyn antibodies. Src-IPs and Fyn-IPs were immunostained with anti–Src-P-Tyr527 antibody, which recognizes the phosphorylated C-terminal sequence of both kinases, and then reprobed with anti-Src or anti-Fyn antibody, respectively. Lyn-IPs were immunostained with anti–Lyn-P-Tyr507 antibody and reprobed with anti-Lyn antibody. The Western blots of Src-IPs are representative of experiments performed with 14 normal samples, 10 PV (JAK2V617F either homozygous or heterozygous), and 18 ET JAK2V617F negative and positive; the Western blots of Fyn-IPs and Lyn-IPs are representative of experiments performed with 4 normal and 4 ET samples. (B) Src kinase was immunoprecipitated from platelet lysates of 5 healthy donors, or 3 AML and 5 MDS patients. Src-IPs were immunostained with anti–Src-P-Tyr527 antibody and then reprobed with anti-Src antibody. (C-D) Platelets, isolated from healthy donors, and PV or ET patients, were incubated with vehicle (lanes 1,3,5) or 100 mU/mL thrombin (lanes 2,4,6) for 1 minute, in the absence (lanes 1-2) or presence of 10μM AG490 (lanes 3-4), or 10μM PP2 (lanes 5-6). Platelets were then lysed in IP-buffer and immunoprecipitated with anti-Src antibody. (C) Src-IPs were analyzed by Western blot with an antibody raised against the Src sequence containing the phospho-Tyr416. Means of densitometric values are reported above the Src-P-Tyr416 bands. Blots were then reprobed with anti-Src antibody. **P < .001 versus Src-P-Tyr416 present in platelets stimulated in the absence of inhibitors. (D) Src-IPs were analyzed for in vitro kinase activity toward the Src-specific peptide substrate cdc2(6-20) as described in “In vitro tyrosine kinase assays.” Samples were subjected to SDS/PAGE and the gels were analyzed for the radioactivity incorporated in the peptide by a Packard Cyclone (columns of the panel). The gels were then blotted and immunostained with anti-Src antibody. Panels C and D are representative of experiments performed with 12 normal samples, 10 PV (JAK2V617F either homozygous or heterozygous), 18 ET JAK2V617F negative or positive. **P < .001 versus resting normal platelets.

Analysis of Src activation state in resting and thrombin-stimulated platelets. (A) Platelets, isolated from healthy donors or PV and ET patients, were lysed in IP-buffer and immunoprecipitated with anti-Src, anti-Fyn, or anti-Lyn antibodies. Src-IPs and Fyn-IPs were immunostained with anti–Src-P-Tyr527 antibody, which recognizes the phosphorylated C-terminal sequence of both kinases, and then reprobed with anti-Src or anti-Fyn antibody, respectively. Lyn-IPs were immunostained with anti–Lyn-P-Tyr507 antibody and reprobed with anti-Lyn antibody. The Western blots of Src-IPs are representative of experiments performed with 14 normal samples, 10 PV (JAK2V617F either homozygous or heterozygous), and 18 ET JAK2V617F negative and positive; the Western blots of Fyn-IPs and Lyn-IPs are representative of experiments performed with 4 normal and 4 ET samples. (B) Src kinase was immunoprecipitated from platelet lysates of 5 healthy donors, or 3 AML and 5 MDS patients. Src-IPs were immunostained with anti–Src-P-Tyr527 antibody and then reprobed with anti-Src antibody. (C-D) Platelets, isolated from healthy donors, and PV or ET patients, were incubated with vehicle (lanes 1,3,5) or 100 mU/mL thrombin (lanes 2,4,6) for 1 minute, in the absence (lanes 1-2) or presence of 10μM AG490 (lanes 3-4), or 10μM PP2 (lanes 5-6). Platelets were then lysed in IP-buffer and immunoprecipitated with anti-Src antibody. (C) Src-IPs were analyzed by Western blot with an antibody raised against the Src sequence containing the phospho-Tyr416. Means of densitometric values are reported above the Src-P-Tyr416 bands. Blots were then reprobed with anti-Src antibody. **P < .001 versus Src-P-Tyr416 present in platelets stimulated in the absence of inhibitors. (D) Src-IPs were analyzed for in vitro kinase activity toward the Src-specific peptide substrate cdc2(6-20) as described in “In vitro tyrosine kinase assays.” Samples were subjected to SDS/PAGE and the gels were analyzed for the radioactivity incorporated in the peptide by a Packard Cyclone (columns of the panel). The gels were then blotted and immunostained with anti-Src antibody. Panels C and D are representative of experiments performed with 12 normal samples, 10 PV (JAK2V617F either homozygous or heterozygous), 18 ET JAK2V617F negative or positive. **P < .001 versus resting normal platelets.

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