Figure 6.
Regions of p150 required for protein binding and cell viability. (A) N-terminal or C-terminal deletion and missense mutant p150 proteins used are schematically represented on the left, and results are summarized on the right. For rescue assay, the p150-conditional knockout cells (1 × 107) were transfected with pApuro-p150-FLAG and its derivatives and cultured in the presence of 0.4 μg/ml puro with or without tet. (a) Numbers of surviving colonies were counted at 10 d after transfection and shown in numerator (+tet) and denominator (−tet), respectively. (b) From several puro-resistant clones in each transfection, one clone expressing almost similar levels of a series of p150-FLAG proteins was chosen and examined for growth capacity with or without tet. In the presence of tet, clones that were dead within three days are indicated with −, and clones that continuously grew for >2 wk are indicated with + in the table. (B) Interaction of p150 and its derivatives with p60, p48, PCNA, and HP1-γ. Stable p150-conditional knockout cell lines, expressing p150-FLAG protein and its derivatives, were used. Cell extracts were prepared from indicated clones (top lanes) at 24 h after the addition of tet and immunoprecipitated with anti-FLAG antibody-coupled beads (M2; Sigma-Aldrich). Input samples before the immunoprecipitation (top; corresponding to 8% of the materials used) and immunoprecipitated samples (bottom) were resolved in 8 or 12.5% SDS-PAGE and then analyzed by Western blotting, by using antibodies for FLAG, p60, p48, PCNA, and HP1-γ. The results obtained are schematically indicated with + and − in the table in A. (C) Growth curves of p150-conditional knockout cell lines expressing FLAG (as a control), wild-type p150-FLAG protein, and its derivatives. Indicated cells were cultured in the presence (+tet) or absence (−tet) of tet. Cells were inoculated in 24-well dish with 1 ml of medium at 2 × 105 cells/ml at day 0. At indicated times, cells were counted and passaged by diluting culture to be 2 × 105 cells/ml.