To obtain the ratio intensities, regions were drawn manually on the tubulin channel and transferred to a phosphorylated ABK (pABK) channel.
To examine if the observed results were due to mislocalizing or inhibiting ABK, levels of active phosphorylated ABK (pABK) at midzones were quantified in control and AAK-inhibited conditions.
This indicates that substrates are less phosphorylated by the midzone aurora kinase activity gradient when AAK activity is inhibited in S2 cells--despite having normal levels (or even higher levels per MT) of midzone pABK. This finding is consistent with previous work that identified transforming acidic coiled-coil containing protein 3 (TACC3) and p150Glued as midzone AAK substrates in human cells that were less phosphorylated and mislocalized, respectively, following AAK inhibition (Lioutas and Vernos.
Subito depletion resulted in a substantial reduction in midzone levels of pABK (Fig.