Identification of the <i>Mib1</i>-interacting domain (MID) in the Notch ligand DeltaD.

2015-05-28T03:35:38Z (GMT) by Gregory Palardy Ajay B. Chitnis
<p>(A) DeltaD deletion constructs and point mutations. Asterisks represent relative positions of NN and KNxNKK motifs. (B) Mib1 does not interact effectively with DeltaD ∆B-D. Myc-Mib1 was co-immunoprecipitated with full-length DeltaD and truncation mutants (∆D, ∆C-D, ∆B-D) using zdd2 antibody (Ab) and detected with anti-Myc Ab. (C) ∆B-D is not effectively ubiquitylated by Mib1. Full-length and DeltaD truncation mutants, co-transfected with HA-ubiquitin (HA-Ub), with and without Myc-Mib1, were immunoprecipitated with zdd2 Ab and immunoblotted with anti-HA Ab to detect ubiquitylated DeltaD. (D) Delta ∆A (∆A) and Delta ∆B (∆B) interact poorly with Mib1. HA-tagged DeltaD and deletion constructs co-transfected with and without Myc-Mib1 are immunoprecipitated with anti-Myc Ab and detected with anti-HA Ab. Relative density of IP anti-Myc band normalized to lysate anti-HA band. (E) ∆B is not effectively ubiquitylated by Mib1. DeltaD-HA and deletion constructs are immunoprecipitated with anti-HA Ab to detect total ubiquitylated DeltaD with and without Myc-Mib1.</p>