Additional file 6.
Co-IP verification of yeast two hybrid interacting pairs identified in AD-wrmcDNA and AD-ORFeome screens using a B2 tailed construct. Additional file 6 is a figure showing the Co-IP verification of yeast two hybrid interacting pairs identified in AD-wrmcDNA and AD-ORFeome screens using a B2 tailed construct. (A) Schematic representation of PDZ domains carrying N-terminal 3XHA epitope tag and of their interacting protein, ending with the B2 tail, carrying N-terminal MYC epitope. (B) Each pair of constructs to be tested was co-expressed in 293T cells and co-IP was performed using cellular lysates subjected to precipitation with anti-HA sepharose. 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 and color code is used to indicate the outcome (purple: interaction tested positive, grey: no interaction and yellow: inconclusive as one or both partners are not expressed). (C) Each ORF used in above co-IP experiment was also subjected to co-transfection and co-immunoprecipitation with empty pDEST-CMV-3xHA vector to serve as a negative control for the binding assay. Detection and analysis were performed as above.
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Lenfant et al. BMC Genomics 2010 11:671 doi:10.1186/1471-2164-11-671