Figure 3. A pericentrin mutant devoid of CEP215 interaction.

(A) HEK293T cells transfected with FLAG-tagged GFP, PCNT (WT), PCNT^1235,1241AA (AA) and PCNT^{Δ2390–2406} (Δ17) were treated with STLC for 16 h to synchronize the cells at mitosis. The mitotic cell lysates were subjected to immunoprecipitation with FLAG resin followed by immunoblot analysis with CEP215 and FLAG antibodies. (B–D) The pericentrin-depleted HeLa cells were rescued with FLAG-GFP-tagged PCNT (WT), PCNT^1235,1241AA (AA) and PCNT^{Δ2390–2406} (Δ17). The cells were treated with RO3306 for 16 h and subsequently removed for 40 min to allow accumulation of mitotic cells. The cells were coimmunostained with CEP215 (red) and FLAG (green) antibodies. Scale bar, 10 µm. The intensities of ectopic pericentrin (C) and endogenous CEP215 (D) at the spindle poles were quantified in more than 40 cells per group in three independent experiments. Error bars, SEM. The paired t-test was performed with p value indicated.