Changes in the expression and phosphorylation of Bcl-2 family members after deprivation or stimulation of cytokines in WtFLT3-32D cells. (A) Phosphorylation of FLT3 and STAT5a in WtFLT3-32D cells. Cells were washed 3 times with RPMI 1640 containing 10% FCS and resuspended with the same medium in the presence or absence of FLT3 ligand (FL, 50 ng/mL). After 0, 12, and 24 hours, each cell lysate was immunoprecipitated by anti-FLT3 antibody or anti-STAT5a antibody, and the immunocomplex was analyzed by immunoblotting with antiphosphotyrosine antibody 4G10. (B) The expression levels of Bcl-2 family members in WtFLT3-32D cells. After 0, 12, 24, and 36 hours, each sample was lysed and Western blotting analysis was performed with anti–Bcl-2, Bcl-XL, Bax, Bak, and Mcl-1 antibodies. (C) Immunoprecipitation by anti-Bad antibody at each time and immunoblotting by antiphospho Bad (Ser112) and antiphospho Bad (Ser136) specific antibodies were carried out.
