Figure 3

Co-IP verification of yeast two hybrid interacting pairs identified in AD-wrmcDNA screen. (A) Schematic representation of PDZ domains carrying N-terminal 3XHA epitope tag and of their full-length interaction partner terminated with a stop codon (ORF*) carrying N-terminal MYC epitope. Each corresponding pair of constructs to be tested was co-expressed in 293T cells and cellular lysates were subjected to precipitation with anti-HA Sepharose. (B) Presence of interacting protein upon precipitation was revealed by western blotting using anti-MYC serum. For each IP performed three panels are presented. Upper panel: IP reaction probed upon resolution on SDS-PAGE and blotting with anti-HA antibody detecting HA-PDZ domain; Middle panel: the same IP reaction probed with anti-MYC serum detecting ORF (MYC:ORF*); lower panel: detection of expression of each ORF by probing total crude cellular extracts (input) with anti-MYC serum. Table summarizes the interaction pairs tested.