(A) Expression of caspase-3 zymogen protein (32 kD) in human CLL cells at 4 and 24 hours after incubation with medium alone or 0.18 or 0.33 μmol/L of flavopiridol. The cells were obtained from CLL patients after obtaining informed consent, isolated, and cultured at 5 × 106/mL in medium or flavopiridol (0.18 or 0.33 μmol/L). Cell lysates were prepared and protein concentration was quantified using the BCA method (Pierce). Fifteen micrograms of protein/lane from the CLL cell lysates was loaded onto a 14% SDS-PAGE gel and electrophoresed. The caspase-3 protein was detected using an anti–caspase-3 polyclonal antibody (Santa Cruz). Lane equivalent loading was certified by assessment with Fast Green Staining (not shown). (B) Expression of caspase-3 zymogen protein (32 kD; identified by a solid arrow) and cleavage product (20 kD; identified by an open arrow) in human CLL cells at 4 and 24 hours after incubation with medium alone or 0.18 or 0.33 μmol/L of flavopiridol. The cells were obtained from CLL patients after obtaining informed consent, isolated, and cultured at 5 × 106/mL in medium alone or 0.18 or 0.33 μmol/L of flavopiridol. Cell lysates were prepared and protein concentration was quantified using the BCA method (Pierce). Seventy-five micrograms of protein/lane from the CLL cell lysates was loaded onto a 14% SDS-PAGE gel and electrophoresed. The caspase-3 protein was detected using an anti–caspase-3 polyclonal antibody (Santa Cruz). Overexposure of the nitrocellulose gel was necessary due to the instability of the cleavage product. Lane equivalent loading was certified by assessment with Fast Green Staining (not shown).
