Megakaryoblast Culture and Signaling

For megakaryoblast culture, BM lineage negative cells from control (Jak2^+/+), heterozygous (Jak2^VF/+), hemizygous (Jak2^VF/−) and homozygous Jak2V617F (Jak2^VF/VF) mice were enriched using a lineage depletion kit (Miltenyi Biotec, Auburn, CA, USA) and were cultured in StemPro medium containing 20 ng/ml stem cell factor (SCF) and 50 ng/ml thrombopoietin (TPO) for 4–5 days as previously described.^{16 (link)} Primary erythroblasts were generated from the BM as previously described.^{10 (link)} For signaling studies, megakaryoblasts or erythroblasts were starved for 6 hr in IMDM medium containing 0.5% BSA at 37°C and cell lysates were prepared in radioimmunoprecipitation assay (RIPA) buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS, 2mM Na₃VO₄, 5mM NaF, 100 µg/ml PMSF and protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO, USA]). Immunoblotting was performed using phospho-specific antibodies against Stat5, Akt, or Erk1/2 (Cell signaling Technology, MA), or antibodies against total Jak2, Stat5, Akt or Erk2 (Cell signaling Technology, MA or Santa Cruz Biotechnology, CA). β-actin was used as a loading control.