Temporal regulation of FF1220 condensin I– and FF1154 PRC1-binding mutants of KIF4A. (A) FLAG-tagged KIF4A WT and mutant complexes were isolated from cells progressing from metaphase into anaphase (KIF4A IP). The temporal association of KIF4A to condensin I and II and PRC1 was followed over time using Western blot. (B and C) Relative KIF4A association to condensin I (B) and PRC1 (C) was measured as a function of time and the mean plotted in the line graphs. Error bars indicate the SD for n = 2. (D) HeLa cells depleted of endogenous KIF4A and expressing fluorescent protein tagged KIF4A WT, FF1154AA, ΔCRD, or FF1220AA were imaged undergoing mitosis. Representative maximum projections are shown.