FIGURE 5.
Pin1 is involved in p27Kip1 polyubiquitination. A, in vivo ubiquitination (Ub) assay of FLAG-p27Kip1 in Pin1-WT and -KO MEFs. At 24 h following transfection, cells were treated with 10 μm MG132 to inhibit protein degradation. After 3 h of MG132 treatment, cells were harvested, and lysates were then incubated with FLAG-M2. Western blots were carried out using anti-FLAG or anti-ubiquitin antibody. IP, immunoprecipitation; KO, knock-out. B, reciprocal experiments were carried out as in A, after FLAG beads pulldown, and the beads were cooked at 95 °C for 5 min, and Western blots were done by blotting with either anti-p27Kip1 or anti-ubiquitin antibody. C, effects of Pin1 on co-immunoprecipitation of HA-Cks1 and FLAG-p27Kip1. Immunoblotting analysis of HA-Cks1 bound to FLAG-p27Kip1 in the presence of different concentrations of recombinant Pin1 protein are shown. D, co-immunoprecipitation of HA-Cks1 and FLAG-p27Kip1 in the presence or absence of Pin1. Immunoblotting analysis of HA-Cks1 bound to FLAG-p27Kip1 in Pin1-WT and -KO MEFs, respectively, is shown.