Anti total akt 9272

Ba/F3 was stably transduced with Flag-wild-type and mutated GNB2 (p.G77R) (pMIG vector) as described previously [23 (link)]. Protein levels were evaluated by immunoblot. Cells were lysed, subjected to SDS-PAGE, and transferred to PVDF membrane (Millipore). Each blot was incubated with anti-Flag antibody (#1E6, Wako Pure Chemicals) or anti-Actin antibody (#I-19, Santa Cruz Biotechnology), and its signal was visualized with Immobilon Western Chemiluminescent HRP Substrate (Millipore). Phosphorylation profile in the downstream signaling pathway of GNB2 was assessed by immunoblot with anti-total AKT (#9272) and anti-pAKT (#9271) antibodies purchased from Cell Signaling. To investigate an effect of GNB2 on leukemogenesis, Ba/F3 cells transduced with wild-type GNB2, GNB2 mutant, and mock as above, were transplanted in immunodeficient mice. To assess GNB2-associated leukemic involvement, 2 × 10⁶ cells per mouse were intravenously transplanted into NOD/SCID/γc null (NOG) mice (N = 21 in total) after 2.5 Gy irradiation. GFP positivity was measured in bone marrow cells. To further study GNB2-associated tumorigenesis, 1 × 10⁷ cells per experiment were subcutaneously injected into nude mice (N = 30 in total) and the tumor size was measured every seven days.