Polo-Like Kinase 1 Directs Assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF Complex to Initiate Cleavage Furrow Formation (original) (raw)
Figure 4
Phosphorylation of HsCyk-4 is necessary for Ect2 association.
(A) The indicated stable cell lines transfected with siRNA to deplete endogenous HsCyk-4 were fixed and stained with Ect2 and GFP antibodies, and DNA was stained with DAPI. Percentages indicate the fraction of anaphase cells displaying “positive” Ect2 central spindle localization (_n_≥90 cells) as defined in Materials and Methods. (B) The indicated stable cell lines transfected with siRNA to deplete endogenous HsCyk-4 were synchronized with nocodazole, at which point 22.5 µM purvalanol A was added to the cells. Lysates and Ect2-BRCT–bound fractions were separated by SDS-PAGE and probed with antibodies to HsCyk-4, GFP, and α-tubulin. Lysate represents approximately 5% of input. (C) The indicated stable cell lines transfected with siRNA to deplete endogenous HsCyk-4 were fixed and stained with antibodies to RhoA and GFP, and DNA was stained with DAPI.