Anti jak2 antibody

The JAK kinase assay was performed using the HTScan JAK2 Kinase Assay Kit (Cell Signaling Technology, Danvers, MA, USA) and streptavidin-coated 96-well plates (SigmaAldrich, Saint Louis, MO, USA). HEK293 cells were transfected with plasmids containing Flag-JAK2 or Flag-JH1 for 24 h and then harvested with lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 0.15% Triton X-100, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM NaF, with complete protease inhibitor cocktail) on ice for 30 min. The cell lysates were immunoprecipitated with anti-JAK2 antibody (#sc34479, Santa Cruz Biotechnology, Saint Louis, MO, USA) overnight at 4 °C. Then, 1:10 (v⁄v) Protein A⁄G Plus Agarose (Santa Cruz Biotechnology, Saint Louis, MO, USA) was added to the lysates at 4 °C overnight. Samples were washed three times and resuspended using kinase reaction buffer (60 mM HEPES, pH 7.5, 5 mM MgCl₂, 5 mM MnCl₂, 25 µM Na₃VO₄, 200 µM ATP). Samples were treated with B6, AZD1480, or DMSO for 30 min at 37 °C before incubation with biotinylated FLT3 (Cell Signaling Technology, Danvers, MA, USA) for 1 h at 37 °C. Samples were then processed following the protocol for the HTScan JAK2 Kinase Assay Kit [24 (link)].