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Characterization of caspase-8L: a novel isoform of caspase-8 that behaves as an inhibitor of the caspase cascade by Daisuke Himeji, Takahiko Horiuchi, Hiroshi Tsukamoto, Kenshi Hayashi, Takeshi Watanabe, and Mine Harada Blood Volume 99(11):4070-4078 June 1, 2002 ©2002 by American Society of Hematology Molecular cloning and deduced amino acid sequences of caspase-8L.(A) Identification of caspase-8L, -8a, and -8b in human PBLs by RT-PCR analysis. Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology RT-PCR analysis of the expression of caspase-8a, -8b, and -8L mRNA in normal human tissues and tumor cell lines.(A) Complementary DNAs of selected human normal tissues (obtained from CLONTECH) were amplified by PCR with primers that detect caspase-8a, -8b, ... Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology RNase protection assay in human PBLs.An RNase protection assay was performed as described. Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology Expression analysis of caspase-8L protein in human PBLs.(A) To test an anti–human caspase-8 polyclonal antibody directed against the N-terminus of caspase-8 (BD Pharmingen) for its capacity to detect caspase-8L, in vitro–translated caspase-8a (lanes 1 and 3... Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology Cell death mediation by caspase-8a and -8L and inhibitory role of caspase-8L in MCF-7 cell lines.(A) MCF-7 cells were transiently transfected with 1.5 μg β-galactosidase expression vector (pcDNA3.1/His/LacZ, Invitrogen) plus 1.0 μg empty vector (pcDNA3.1/Hi... Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology Flow cytometric analysis of Fas-induced cell death and colorimetric analysis of Fas-induced caspase-8 catalytic activity in caspase-8L–transfected Jurkat cells.(A,B) Empty vector– or caspase-8L–transfected Jurkat cells and wild-type Jurkat cells were untrea... Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology In vitro interaction of caspase-8L with FADD and caspase-8a and the inhibition of the binding of caspase-8a to FADD by caspase-8L.HS1, caspase-8a, and caspase-8L fused at its N-terminus with the polyhistidine epitope (His) were generated by in vitro transla... Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology In vivo interaction of caspase-8L with FADD in MCF-7 cell lines.MCF-7 cells (2 × 107 cells) were transiently transfected with 4 μg empty vector (pcDNA3.1/HisA, Invitrogen) and His-tagged caspase-8L expression vector using LipofectAMINE (Life Technologies) a... Daisuke Himeji et al. Blood 2002;99:4070-4078 ©2002 by American Society of Hematology
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