American Society of Hematology

Fig. 1.

$Fig. 1. Immunoblot analysis of procaspase-8 activation in TRAIL-treated myeloma cells. / The myeloma cells were incubated with 1 μg/mL leucine-zipper TRAIL (Immunex, Seattle, WA) and harvested at 30 and 60 minutes following incubation. The proportion of cells undergoing apoptosis, shown by the percentage in parentheses at 60 minutes, was determined by annexin-V staining. Cytosolic protein fractions (25 μg) were separated on 4% to 16% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose, and probed with a caspase-8–specific goat polyclonal antibody C-20 (Santa Cruz, Santa Cruz, CA). Proteins were visualized by enhanced chemiluminescence (ECL). Membranes were stripped and reprobed with monoclonal antibody to α-tubulin (Sigma, St Louis, MO). The sensitive myeloma cell lines RPMI 8226, LP-1, and OPM-2 all show early cleavage of procaspase-8 with generation of the intermediate p43/41 product (*) and the active P18 form. Resistant cell lines NCI H929 and U266 demonstrate delayed procaspase-8 cleavage with minimal P18 generation.$

Immunoblot analysis of procaspase-8 activation in TRAIL-treated myeloma cells.

The myeloma cells were incubated with 1 μg/mL leucine-zipper TRAIL (Immunex, Seattle, WA) and harvested at 30 and 60 minutes following incubation. The proportion of cells undergoing apoptosis, shown by the percentage in parentheses at 60 minutes, was determined by annexin-V staining. Cytosolic protein fractions (25 μg) were separated on 4% to 16% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose, and probed with a caspase-8–specific goat polyclonal antibody C-20 (Santa Cruz, Santa Cruz, CA). Proteins were visualized by enhanced chemiluminescence (ECL). Membranes were stripped and reprobed with monoclonal antibody to α-tubulin (Sigma, St Louis, MO). The sensitive myeloma cell lines RPMI 8226, LP-1, and OPM-2 all show early cleavage of procaspase-8 with generation of the intermediate p43/41 product (*) and the active P18 form. Resistant cell lines NCI H929 and U266 demonstrate delayed procaspase-8 cleavage with minimal P18 generation.

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