Identification of critical residues in the Mib1-Interacting Domain (MID).
(A) Conserved amino acids (pink shading) in the putative Mib1-Interacting Domain in Delta ligands. (B) KK and NN/KK mutants do not significantly interact with mind bomb. DeltaD-HA, ∆B and point mutation (NN, KK and NN/KK) constructs are co-immunoprecipitated with Myc-Mib1 using anti-Myc Ab and detected with anti-HA Ab. (C) KK and NN/KK mutants are not significantly ubiquitylated by Mib1. DeltaD-HA, ∆B and point mutation constructs co-transfected with Flag-Ubiquitin (Flag-Ub) with and without Myc-Mib1 are immunoprecipitated with anti-HA Ab and detected with Anti-Flag Ab to assay ubiquitylation of full length and mutant forms of DeltaD.