FIGURE 2.

Aft1 does not impact the kinetochore localization and protein interactions of Iml3. A, presence or absence of Aft1 does not impact the purification of Iml3-TAP. Western blot analysis of Iml3-TAP purified by ChIP (IP) and whole-cell extract (WCE). B, Iml3 localization to the centromere is not affected by Aft1. Traditional ChIP performed using untagged, Iml3-TAP, and Iml3-TAP aft1Δ strains. Total or immunoprecipitated DNA was subjected to multiplex PCR amplification using primers specific to the centromere of chromosome 3 (CEN3) and the negative control FET3. C, Iml3-Ctf19 co-purification is not affected by Aft1. Anti-TAP immunoprecipitations were performed with strains containing different combinations of TAP-tagged Iml3 and Myc-tagged Ctf19 in the presence or absence of Aft1. D, Iml3-Chl4 interaction is not affected by Aft1. Anti-HA immunoprecipitations were performed with strains containing different combinations of HA-tagged Iml3 and Myc-tagged Chl4 in the presence or absence of Aft1. For all immunoprecipitations, 50% of the eluate was loaded in the TAP or HA blot, whereas 50% of the eluate was loaded in the Myc-blot.