Figure 3. MTMR4 and Nedd4 binding is mediated through a PY motif–WW domain interaction.

GST-fusion proteins of the area encompassing the PY motif of hMTMR4 (GST-hMTMR4 PY) and of a non-PY-containing proline-rich region of hMTMR4 (GST-hMTMR4 PRO) as control were generated in bacteria as described in the Experimental section. Samples of 0.3 μg of soluble and GST–PY and GST–Pro were incubated with 0.5 μg of His₆-tagged hNedd4–WWI, –WWII, –WWIII or –WWIV immobilized on Ni²⁺-nitrilotriacetate beads (His-hNedd4 WW domain I–IV) or His₆–beads alone (His). Following thorough washes of the beads, bound proteins were eluted with sample buffer, separated by SDS/PAGE, and Western blotting was performed with anti-GST and anti-His antibodies. Molecular masses are indicated in kDa.