JCS -- Shearwin-Whyatt et al. 117 (16): 3679 Figure IG1

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Fig. 1. N4WBP5A interacts with Nedd4 family members. (A) The interaction between N4WBP5A and the WW domains of Nedd4 is mediated via PY motifs 1 and 2. COS cells were transfected with vector (–), wild-type pcDNA3-N4WBP5A-FLAG (WT), or pcDNA3-N4WBP5A-FLAG in which PY motif 1 was mutated (Y56A; PY1) or PY motif 2 was mutated (Y82A; PY2), or pcDNA3-N4WBP5A-FLAG-PY1,2 (PY1,2). FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and immunoprecipitates subjected to SDS-PAGE and blotted. The blot was probed with a ³²P-labelled GST-Nedd4 protein containing WW domains 1-3 of mouse Nedd4. Upper panel: anti-FLAG western blot showing the relative amount of immunoprecipitated FLAG-tagged proteins. Lower panel: far-western of the above blot probed with ³²P-labelled Nedd4 GST-WW1-3. (B) The interaction between N4WBP5A and Nedd4 is mediated via WW domains 2 and 3. COS cells were transfected with wild-type pcDNA3-N4WBP5A-FLAG (WT), or pcDNA3-N4WBP5A-FLAG in which both PY motifs 1 and 2 were mutated (Y56A, Y82A; PY1,2). FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and immunoprecipitates subjected to SDS-PAGE and blotted. The blot was probed with a ³²P-labelled Nedd4-GST protein containing WW domain 1, 2 or 3 of Nedd4. Upper panel: anti-FLAG western blot showing the relative amount of immunoprecipitated FLAG-tagged proteins. Lower panel: far-western of the above blot probed with ³²P-labelled Nedd4 GST-WW1, GST-WW2 or GST-WW3. (C) N4WBP5A interacts with the WW domains of Nedd4 family proteins. Escherichia coli extracts expressing various GST-WW proteins of the human genes NEDD4, NEDD4-2, KIAA0332, SMURF1, WWP2 and AIP-4 or the mouse gene Itch were blotted and probed with a ³²P-labelled GST-N4WBP5A protein comprising the amino terminal 136 amino acids of N4WBP5A, containing the PY motifs, fused to the carboxyl terminal of GST. Upper panel: Coomassie-stained gel of GST WW domain fusion proteins. Lower panel: far-western blot of a duplicate gel blotted and probed with the ³²P-labelled GST-N4WBP5A protein.