Figure 5. MTMR4 ubiquitination is dependent on an intact PY motif.

(A) HeLa cell lysate transfected with HA-tagged hMTMR4 or HA-tagged hMTMR4 PY motif mutant, together with hNedd4 or untransfected lysate (untx) were subjected to immunoprecipitation (I.P.) with anti-MTMR4 antibodies. Beads were washed and separated by SDS/PAGE with total lysate and then subjected to Western blotting with anti-ubiquitin (Ub) antibodies to detect ubiquitinated MTMR4. Blots were re-probed with anti-HA antibodies to detect transfected hMTMR4 and then stripped and re-probed with anti-Nedd4 antibodies to detect transfected Nedd4. Aliquots of whole-cell lysate were subjected to immunoblotting with anti-HA and anti-Nedd4 antibodies. Blots were stripped and re-probed with anti-α-tubulin antibodies as a loading control. Ubiquitinated MTMR4 is seen as a high-molecular-mass smear (top-left-hand panel) in the hMTMR4 transfected lysate only. Molecular masses are indicated in kDa.