PAX5-JAK2 is phosphorylated and activates STAT proteins. (A) Intracellular phosphoprotein analyses using flow cytometry were performed in JAK2-deficient γ2a cells transfected with the indicated proteins. One of 3 representative experiments is shown. Black lines, empty vector, pIRES2-EGFP; orange lines, PAX5; red lines, JAK2; blue lines, PAX5-JAK2. (B) Western blotting of whole cell lysates of γ2a cells. pc, pcDNA3 empty vector; P-J, PAX5-JAK2; P-J-YY/AA, tyrosine mutant; P-J-K/E, kinase dead mutant (GAPDH ∼ 37 kDa, HA ∼ 60 kDa, pJAK2 ∼ 60 kDa, STATs/pSTATs ∼ 90 kDa). (C) PAX5-JAK2 phosphorylation detected in whole cell lysate of a bone marrow sample of a PAX5-JAK2⁺ patient. −PV, without pervanadate; +PV, treated with phosphatase inhibitor pervanadate; **PAX5-JAK2; *PAX5. (D) pSTAT5 levels in bone marrow cells of patients with PAX5-JAK2⁺ (n = 2), BCR-ABL1⁺ (n = 33) or B-other (n = 43) leukemia were measured by reverse phase protein arrays, and levels were normalized for total protein levels in each cell lysate. (E) Immunofluorescence was performed in HeLa cells transfected with (top) HA-tagged PAX5-JAK2, as well as with the mutated forms (middle) PAX5-JAK2-YY/AA and (bottom) PAX5-JAK2-K/E using an HA (green) and a p-Y1007/8-JAK2 (red) antibody and DAPI (blue) for DNA counterstaining. Images were acquired on a Leica TCS SP5 equipped with an HCX PL APO CS 63.0×1.40 oil objective in sequential scan mode using identical settings for all conditions. White bars indicate 20 µm.