Figure 5

NumbL interacts in a tandem affinity purification approach with subunits of the AP-2 complex. (A) Colloidal coomassie blue stained gradient gel of two subsequent immunoprecipitations from X. laevis embryos injected in both blastomeres with 500 pg of NumbL-CTap or CTap mRNA. The bands representing NumbL and major specifically co-immunoprecipitated proteins (i.e. Xenopus proteins that were identified in the NumbL-CTap, but not in the CTap lane) are indicated on the gel as follows: 1, AP-2 beta 1 subunit, AP-2 alpha 2 subunit; 2, NumbL; 3, AP-2 mu1 subunit. ( B) Table listing the protein identification details, the numbering of the bands refers to the gel image in Figure 5A. The number of peptides sequenced refers to the set of non-redundant peptides that have been assigned to the protein sequence according to the MASCOT MS/MS ion search algorithm. (C-H) Putative AP-2 binding mutants of NumbL are not able to rescue the MO phenotype. Embryos were injected into one blastomere at the two-cell stage with 500 pg of NumbL mRNA and 12.5 ng NumbL MO as indicated in the upper right corner together with β-gal mRNA (75 pg) and N-tubulin expression was analyzed by whole mount in situ hybridization. Shown are stage 15 embryos in a dorsal view, anterior up. The injected site is marked by X-Gal staining (light blue) and is always on the right. The number of embryos (n) and percentage of embryos showing the described phenotype are indicated in the lower right corner. The midline is indicated with a red arrowhead.