Fig. 6

Phosphorylated JIP1 enhances Itch phosphorylation by Cdk5 to promote Notch1-IC ubiquitination. A Western blot analysis of phospho-Itch (p-Itch@T222) and Itch (Itch) in total cell lysate from JIP1 WT and KO neurons (DIV 3). Graph represents quantification of phospho-Itch levels relative to Itch in JIP1 WT and KO (n=4 each). Groups were compared using Student’s t-test and data presented as mean ± SEM. “n” equals the number of animals. ** p < 0.01. B Western blot analysis of in vitro binding assay using bacterially expressed GST or GST fused-p35 incubated with His-JIP1 and/or Itch recombinant proteins at 4 °C for 2 h. GST-tagged proteins were retrieved with GSH beads. The proteins were immunoblotted with Itch and JIP1 antibody. Data representative of three independent experiments. C Immunoblot analysis of CD-1 embryonic cortex lysate (E15.5-16.5) immunoprecipitated with Itch antibody and probed with JIP1 antibody. Data representative of three independent experiments. D Western blot analysis of Itch phosphorylation by Cdk5. Recombinant Itch protein incubated with Cdk5/p35 with or without 20 μM of Roscovitine (Rosco) at 30 °C for 2 h was immunoblotted with phospho-Itch (pItch@T222) and Itch antibody. Data representative of three independent experiments. E Western blot analysis of the contribution of JIP phosphorylation to Itch phosphorylation. Recombinant JIP1 or T205A was incubated with Cdk5/p35 at 30 °C for 1 h followed by the addition of Itch and incubated for 15 min. The mixture was then analyzed by immunoblotting with phospho-Itch and Itch antibody. F Quantification of phospho-Itch levels in E. Multiple comparison was analyzed by one-way ANOVA and data are presented as mean ± SEM from four independent experiments. * p < 0.05. G Wild-type Itch (WT) or Itch mutants (phosphomimic T222D (T222D) or Ala mutant T222A (T222A)) were co-transfected into HEK293A cells with Notch1-IC-Flag and immunoprecipitated with Notch1 antibody. Immunoprecipitated proteins were subjected to western blot analysis with Ubiquitin (UB) antibody. Data representative of three independent experiments