(A) Expression of bcl-2 protein in human CLL cells at 4 and 24 hours after incubation with medium or 0.18 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 μmol/L). Cell lysates were prepared and protein concentration was quantified using the BCA method (Pierce). Two micrograms of protein/lane from the CLL cell lysates was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed. Bcl-2 protein was detected using an anti–bcl-2 monoclonal antibody (Dako). Lane equivalent loading was certified by assessment with Fast Green Staining (not shown). (B) Expression of p27 protein in human CLL cells at 4 and 24 hours after incubation with medium or 0.18 and 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). Five micrograms of protein/lane from the CLL cell lysates was loaded on a 10% SDS-PAGE gel and electrophoresed. The p27 protein was detected using an anti-p27 polyclonal antibody (Oncogene). Lane equivalent loading was certified by assessment with Fast Green Staining (not shown).
