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Protein phosphatase 2A activates the proapoptotic function of BAD in interleukin- 3–dependent lymphoid cells by a mechanism requiring 14-3-3 dissociation by Chi-Wu Chiang, Gregory Harris, Cindy Ellig, Shane C. Masters, Romesh Subramanian, Shirish Shenolikar, Brian E. Wadzinski, and Elizabeth Yang Blood Volume 97(5):1289-1297 March 1, 2001 ©2001 by American Society of Hematology Okadaic acid inhibits BAD dephosphorylation.(A) FL5.12BCL-XL/BAD cells were treated with 0.5 μM OA or dimethyl sulfoxide (DMSO) for 4 hours in the presence (+) or absence (−) of IL-3. Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology PP2A-selective inhibitors block BAD dephosphorylation in vivo.(A) FL5.12BCL-XL/BAD cells were treated with PP1 and PP2A inhibitors calyculin A, fostriecin, and tautomycin in the presence of IL-3 for 3 hours. Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology PP2A activity is significantly inhibited by in vivo OA and fostriecin treatment.(A) Phosphatase activity in lysates of cells treated with 0.5 μM OA (OA) using either 32P-labeled phosphorylase a (upper panel) or 32P-labeled histone H1 (lower panel) as substr... Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology 14-3-3 prevents BAD dephosphorylation in vitro.(A) FL5.12BCL-XL/BAD lysates were prepared in phosphatase buffer A with or without 1% empigen BB (Emp) (upper panel) or 25 μM R18 peptide (lower panel). Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology PP2A-selective inhibitor, but not PP1-specific inhibitor, inhibits BAD dephosphorylation in vitro.(A) FL5.12BCL-XL/BAD lysates were prepared in phosphatase buffer A with or without fostriecin, in the presence or absence of 25 μM R18 peptide, incubated at 30... Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology Serine/threonine phosphatase inhibitor calyculin A rescues FL5.12 cells expressing BAD from apoptosis after IL-3 withdrawal.Cells expressing wild-type BAD (A) or the phosphorylationdefective mutant BAD (B) were treated with 5 nM calyculin A (⋄, ▵) or vehic... Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology Activation of the proapoptotic function of BAD by phosphatase in IL-3–dependent FL5.12 cells is regulated by 14-3-3 binding.In the presence of survival factor IL-3, BAD is phosphorylated and binds 14-3-3. Chi-Wu Chiang et al. Blood 2001;97:1289-1297 ©2001 by American Society of Hematology