p27fs177 is degraded very fast also in cells stably-transfected with the GFP fusion constructs. (a) Clones stably expressing the various p27 mutants. (b) CHX treatment for clones stably expressing p27wt, p27fs177 and p27G177X. Only the exogenous p27 proteins are shown. (c) The p27-unrelated, C-terminal domain of p27fs177 is responsible for the rapid degradation of the protein. The CDK2 expression vectors shown on the right were transfected into MCF7 cells and protein extracts were immunoprecipitated (IP) overnight with anti-GFP antibody. (d) p27fs177 is very rapidly degraded independently from the cell cycle phase. p27fs177 turn-over determination in G1-phase (G1), and S-phase (S) in Clone 9 cells. (e) p27fs177 is in part degraded through the proteasome. Proliferating p27fs177-Clone 9 cells were treated with CHX and epoxomycin (EPOX, +) or DMSO (-) for the indicated times. Untr, untransfected; IB, immunoblot; IP, immunoprecipitation; EPOX, epoxomycin.