Figure 6

Numb5 interacts with cytoskeleton and polarity regulating proteins. (A) Immunoprecipitation assay of cellular proteins interacting with Numb5 using Myc antibody. Lanes represent protein separation in N2a cells expressing LacZ-Myc-His and Numb5-Myc-His. The potential interacting proteins are shown in boxes. (B) Mass spectral sequence analysis of the protein band at approximately 53 kDa. Peptide mapping analysis confirms that the Numb5-interacting protein is vimentin (upper panel). The sequences of peptide fragments generated for vimentin residues from amino acids 391 to 404 (lower panel). (C) Confocal immunofluorescence detection of vimentin protein in N2a cells over-expressing Numb5-GFP. Green shows Numb5 detection in N2a cells infected with Numb5-GFP; red shows vimentin protein expression detected with vimentin antibody and Alexa546 fluorophore; yellow shows the merged image. Scale bar = 7 μm. (D) Western blot detection of IQGAP1 protein in N2a cells over-expressing Numb5-Myc. Numb5 interacts with the polarity and migration regulating protein IQGAP1. Total protein lysates from N2a cells expressing LacZ-Myc-His or Numb5-Myc-His were electrophoretically separated and subjected to Western blotting using IQPAP1 antibody.