American Society of Hematology

Fig. 4.

$Fig. 4. Expression analysis of caspase-8L protein in human PBLs. / (A) To test an anti–human caspase-8 polyclonal antibody directed against the N-terminus of caspase-8 (BD Pharmingen) for its capacity to detect caspase-8L, in vitro–translated caspase-8a (lanes 1 and 3) and -8L (lanes 2 and 4) were analyzed by SDS-PAGE. The reaction mixtures (2 μL for caspase-8a, 4 μL for caspase-8L) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected by HRP-conjugated streptavidin (left column) or by anti–human caspase-8 antibody (right column). They are revealed by Transcend Chemiluminescent Non-Radioactive Translation Detection System (Promega) (left column) or by HRP-conjugated secondary antibody and the ECL reagents (Amersham Pharmacia Biotech). (B) A Western blot analysis of the expression of caspase-8a, -8b, and -8L on resting human PBLs. In vitro–translated caspase-8L (lane 1) and cellular extracts from COS-7 cells transfected with a construct encoding caspase-8L (lane 2), human PBLs (lane 3), and Daudi cells (lane 4) were fractionated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Next, the membrane was probed with anti–human caspase-8 polyclonal antibody (BD Pharmingen) and visualized with secondary antibody as described above. The positions of molecular mass markers (in kilodaltons) are shown on the left in all panels.$

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) and -8L (lanes 2 and 4) were analyzed by SDS-PAGE. The reaction mixtures (2 μL for caspase-8a, 4 μL for caspase-8L) were separated on 10% SDS-PAGE, transferred to nitrocellulose membranes, and detected by HRP-conjugated streptavidin (left column) or by anti–human caspase-8 antibody (right column). They are revealed by Transcend Chemiluminescent Non-Radioactive Translation Detection System (Promega) (left column) or by HRP-conjugated secondary antibody and the ECL reagents (Amersham Pharmacia Biotech). (B) A Western blot analysis of the expression of caspase-8a, -8b, and -8L on resting human PBLs. In vitro–translated caspase-8L (lane 1) and cellular extracts from COS-7 cells transfected with a construct encoding caspase-8L (lane 2), human PBLs (lane 3), and Daudi cells (lane 4) were fractionated by 10% SDS-PAGE and transferred to a nitrocellulose membrane. Next, the membrane was probed with anti–human caspase-8 polyclonal antibody (BD Pharmingen) and visualized with secondary antibody as described above. The positions of molecular mass markers (in kilodaltons) are shown on the left in all panels.

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