American Society of Hematology

Fig. 1.

$Fig. 1. Molecular cloning and deduced amino acid sequences of caspase-8L. / (A) Identification of caspase-8L, -8a, and -8b in human PBLs by RT-PCR analysis. The total RNA from the PBLs of a healthy human individual were reverse transcribed and then amplified by PCR with primers encompassing the entire coding region of caspase-8a and -8b. The resulting RT-PCR products were resolved on 1.2% agarose gel and visualized by ethidium bromide staining. In addition to 2 expected bands (1587 bp and 1542 bp) representing caspase-8a and -8b, a long amplification product, representing caspase-8L, was identified (1723 bp). (B) Deduced amino acid sequence of caspase-8L compared with caspase-8a (GenBank accession number X98172). The DEDs are shaded. Amino acids that were implicated for the catalytic activity of caspase-8a are marked by ● below the alignment. Caspase-8L, a splice variant of caspase-8a, is truncated at amino acid residue 276, including C-terminal 8 amino acids different from caspase-8a (italics). The C-termini of these proteins are denoted by asterisks. (C) Molecular structures of caspase-8a and -8L are shown schematically. The DEDs are denoted by the checked areas. The catalytic cysteine residue and amino acids that were implicated in catalytic activity are indicated by ●. Amino acid residues in the unique C-terminal sequences of caspase-8L are heavily shaded. (D) Western blots of in vitro–translated caspase-8a and -8L. Full-length cDNA fragments encoding caspase-8a and -8L were subcloned into pcDNA3 and in vitro translated as described. The translated products (caspase-8a, 2 μL; caspase-8L, 4μL) were fractionated on 10% SDS-PAGE, transferred to nitrocellulose membrane, and visualized as described. In lane 1, an empty vector (pcDNA3) was in vitro translated and analyzed as a negative control.$

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. The total RNA from the PBLs of a healthy human individual were reverse transcribed and then amplified by PCR with primers encompassing the entire coding region of caspase-8a and -8b. The resulting RT-PCR products were resolved on 1.2% agarose gel and visualized by ethidium bromide staining. In addition to 2 expected bands (1587 bp and 1542 bp) representing caspase-8a and -8b, a long amplification product, representing caspase-8L, was identified (1723 bp). (B) Deduced amino acid sequence of caspase-8L compared with caspase-8a (GenBank accession number X98172). The DEDs are shaded. Amino acids that were implicated for the catalytic activity of caspase-8a are marked by ● below the alignment. Caspase-8L, a splice variant of caspase-8a, is truncated at amino acid residue 276, including C-terminal 8 amino acids different from caspase-8a (italics). The C-termini of these proteins are denoted by asterisks. (C) Molecular structures of caspase-8a and -8L are shown schematically. The DEDs are denoted by the checked areas. The catalytic cysteine residue and amino acids that were implicated in catalytic activity are indicated by ●. Amino acid residues in the unique C-terminal sequences of caspase-8L are heavily shaded. (D) Western blots of in vitro–translated caspase-8a and -8L. Full-length cDNA fragments encoding caspase-8a and -8L were subcloned into pcDNA3 and in vitro translated as described. The translated products (caspase-8a, 2 μL; caspase-8L, 4μL) were fractionated on 10% SDS-PAGE, transferred to nitrocellulose membrane, and visualized as described. In lane 1, an empty vector (pcDNA3) was in vitro translated and analyzed as a negative control.

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