The p27fs177 mutant protein is expressed at low level in vitro due to enhanced degradation. (a) Left: Schematic representation of the constructs used. Right: Steady-state level of the three transfected p27 proteins: GFP-p27wt, -p27fs177 and -p27G177X. (b) The level of p27fs177 does not increase with time post-transfection. p27wt and p27fs177 steady-state levels were analyzed at different times after transfection into MCF7 cells by Western blotting as in A. On the left, p27wt or p27fs177 indicate the transfected proteins. (c) p27fs177 is unstable compared to p27wt. Kinetic of degradation of p27wt, p27fs177 and p27G177× proteins determined after transfection into asynchronously growing MCF7 cells and treatment with cycloheximide (CHX) for the indicated times. (d) p27fs177 has a half-life of about 1 hr. Transfected MCF7 cells were incubated with CHX for shorter time compared to panel C. (e) Live cell imaging of MCF7 cells transfected with p27wt or p27fs177 and treated for the indicated times with CHX confirms the rapid degradation of p27fs177. Wt, wild-type; Untr, untransfected; IB, immunoblot; Exo, exougenous; Endo, endogenous; GFP, green fluorescent protein; CHX, cycloheximide.