Differential binding of phosphodynein and dephosphodynein. (A) Blot overlay assays demonstrate binding of dephospho-ICs to p150^Glued but reduced binding of phospho-ICs to p150^Glued coupled with enhanced binding to an ∼90-kD protein that co-migrates with zw10. Blot overlays of zw10 immunoprecipitates with phospho-ICs display this interaction in parallel. The binding of ICs to p150^Glued changes from an intensity of 83.02% of ICs to 55.39% of ICs after phosphorylation (P = 0.0111), whereas binding of ICs to zw10 increases from 31.12% of ICs to 96.22% of ICs after IC phosphorylation (P = 0.0004). The numbers to the left of the blots indicate the measurement in kilodaltons. (B) Binding of dephospho-ICs (de-P0₄-DIC), T89A and T89D mutant ICs, and phospho-ICs (PO₄-DIC) to zw10 revealed enhanced binding for only phospho-ICs (∼100% increase). Differences between T89A/T89D mutants and the dephospho-ICs were not significant, whereas the difference between dephospho- and phospho-ICs was significant (P = 0.012). Black lines indicate that intervening lanes have been spliced out. (C) Western blot (WB) analysis demonstrating shRNA-based differences. shRNA-based depletion of zw10 and p150^Glued revealed an ∼60% decrease in zw10 signal after zw10 depletion but no effect on p150^Glued. shRNA-based depletion of p150^Glued resulted in depletion of p150^Glued but had no effect on zw10. (D) IFM analysis of PT89-dynein in mitotic cells revealed loss of PT89 signal (red) in cells treated with zw10 shRNA constructs encoding GFP. PT89 signal intensity is presented using a color gradient representing intensities of 0–2,300 intensity units. The number of kinetochores displaying control mean intensity are compared for control and zw10-depleted cells (P < 0.05). The circled area shows a cell expressing zw10 shRNA. Error bars represent SD. Bar, 5 μm.