Itch directly interacts with Glis3 through its WW-domains.

<p>A. HEK293T cells were transfected with FLAG-Glis3 and Myc empty vector, Itch, Itch-Δ-C2, Itch-Δ-HECT, or Itch-WW_I containing only the four WW-domains. Co-immunoprecipitation was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131303#pone.0131303.g002" target="_blank">Fig 2A–2C</a>. B. FLAG-Glis3 or the <i>PY</i>^<i>461</i> mutant and Myc Empty or Myc-Itch were transcribed and translated <i>in vitro</i> and an <i>in vitro</i> pulldown assay was performed using anti-Myc antibody as described in Methods and Materials. G. Schematic showing the interaction between Itch with the <i>PY</i>^<i>461</i> motif of Glis3.