Figure 4
p27Kip1 Interacts with cyclin F. (a) Cells received Dox for 24 h. Cell extracts were immunoblotted with antibody to cyclin F. (b) p27K (clones 2 and 8) and p27CK cells received Dox for 24 h. Anti-HA immunoprecipitates were immunoblotted with antibody to cyclin F or HA. Input was 0.6% of total protein used for immunoprecipitation. Asterisk denotes non-specific band. (c) Parental (Par), p27WT, p27C, p27K and p27CK cells received Dox for 24 h. Anti-HA immunoprecipitates were immunoblotted with antibody to cyclin F. (d) Skp2 siRNA (SK) or non-targeting (scrambled) siRNA (NT) (40 nM, Thermo Scientific Dharmacon, Lafayette, CO, USA) was introduced into HEK 293T cells using Dharmafect I (Dharmacon). Cells were harvested 48 h after transfection. Cell extracts were incubated with preimmune serum (PI) or antibody to p27Kip1, and precipitated material was immunoblotted with antibody to cyclin F, Skp2 or p27Kip1. Input was 0.4% of total protein used for immunoprecipitation. Actin is shown as a loading control.
