Affinity labeling of etoposide-treated HL-60 and K562 cytosol and nuclei. (A) Aliquots containing 37 μg of cytosolic protein prepared from HL-60 (lanes 2 through 7) or K562 (lanes 8 through 11) cells treated with 68 μmol/L etoposide for the indicated length of time were reacted with z-EK(bio)D-aomk, subjected to SDS-PAGE, and reacted with peroxidase-coupled streptavidin. Additional experiments (see B) indicate that bands detected in 0-hour samples bind streptavidin in the absence of reaction with z-EK(bio)D-aomk. Lane 1, apoptosis-inducing S/M extract prepared from DU249 chicken hepatoma cells.24 53 (B) z-EK(bio)D-aomk labeling of cytosol and nuclei from HL-60 cells and K562 cells treated with 68 μmol/L etoposide for the indicated length of time. Samples containing cytosol or nuclei from 3 × 106 cells were incubated with 1 μmol/L z-EK(bio)D-aomk for 1 hour, then subjected to SDS-PAGE and blotting as described in the legend for (A). Lanes marked 0* contain nuclei that were subjected to SDS-PAGE without z-EK(bio)D-aomk treatment, thereby revealing the streptavidin-binding polypeptides that are normal constituents of these nuclei.
