Immunoblotting-assisted assessment of JAK/STAT and PI3K/Akt/mTOR signaling in myeloproliferative neoplasms CD34+ stem cells.

Philadelphia-negative myeloproliferative neoplasms (pH-MPNs) origin from the clonal expansion of hematopoietic stem cells with acquired mutations leading to uncontrolled proliferation of differentiated myeloid cells. The main entities of Ph-MPNs are represented by Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF) that are characterized by microvascular disorders, thrombosis and bleeding, splenomegaly secondary to extramedullary hematopoiesis, various degree of bone marrow fibrosis and a progressive risk of leukemic transformation. Somatic mutations in myeloid genes including JAK2, CALR, and MPL cause the constitutive activation of the Janus Kinase 2 (JAK)/signal transducer and activator of transcription (STAT) pathway that confers proliferative and differentiative advantage to mutated hematopoietic progenitors and ultimately drives the development of a Ph-MPNs phenotype. Beyond the JAK/STAT axis, a wide number of intracellular signaling pathways were found deregulated in Ph-MPNs including the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) constitutive activation. In this chapter, we provide a detailed protocol for the immunoblotting assisted assessment of Ph-MPNs pathways activation. This protocol can be easily adapted to study protein expression and phosphorylation of hematopoietic stem progenitors and differentiated cell lineages.