Figure 1
Generation of p27KO and mutant p27 3T3 mouse fibroblasts. (a) Schematic representation of the experimental workflow used to generate 3T3 p27 mutant clones. Briefly, fibroblasts were harvested from p27KO mouse embryos, immortalized using the 3T3 protocol and transduced with p27WT or mutants of interest. (b) Schematic representation of p27WT protein structure and the two mutants of interest, generated by point mutations in the cyclin- CDK- binding domains (p27CK−) or in the nuclear localization signal (p27KR). (c) Western Blot analysis of immunoprecipitated (IP) proteins and lysates harvested from indicated 3T3 cell clones. CTR represents p27KO fibroblasts; WT, KR, CK− represent respectively the p27WT, KR and CK− represents (both human and mouse) transduced-fibroblasts. (d) Immunofluorescence analysis of above described clones, showing p27 expression and localization. Nuclei were stained with DAPI. (e) Growth curve analysis of above described clones. Cells were plated in 6-well plates in duplicates and counted every day, for 4 consecutive days. Data are expressed as fold increase over the number of plated cells (1 × 105) with standard deviation. MEF, mouse embryo fibroblasts; BD, binding domain; NLS, nuclear localization signal; NES, nuclear export signal.
