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. 2009 May 26;7(5):e1000110. doi: 10.1371/journal.pbio.1000110

Figure 4. Phosphorylation of HsCyk-4 is necessary for Ect2 association.

Figure 4

(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.