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Fig. 2. N4WBP5A is ubiquitinated by Nedd4 and Nedd4-2. (A) As indicated above the lanes, immunoprecipitations were carried out from 293T cells transfected with pcDNA3-N4WBP5A-FLAG (5A WT), or pcDNA3-N4WBP5A lysine mutant (5A K4) and vector or HA-ubiquitin (Ub-HA) using a control antibody (lane 1) or anti-N4WBP5A antibody (lanes 2-7). In lanes 5 and 7, cells were treated with MG132 for 4 hours before lysis. Upper panel: anti-N4WBP5A western blot showing immunoprecipitated N4WBP5A proteins. Lower panel: anti-HA western blot showing ubiquitinated immunoprecipitated proteins. (B) Immunoprecipitations were carried out from 293T cells transfected with pcDNA3-N4WBP5A (5A WT), or pcDNA3-N4WBP5A-PY1,2 (5A PY1,2), HA-ubiquitin (Ub-HA), and increasing amounts of Nedd4 (N4, 0.5 and 1.0 µg), Nedd4 cysteine mutant (N4 cys, 1.0 µg), Nedd4-2 (N4-2, 0.5 and 1.0 µg) or vector (lane 1) using the anti-N4WBP5A antibody. Upper panel: anti-N4WBP5A western blot showing immunoprecipitated N4WBP5A proteins. Lower panel: anti-HA western blot showing ubiquitinated immunoprecipitated proteins.





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