Detection of Human Hakai by Western Blot of Immunoprecipitates.  Samples: Whole cell lysate (1 mg for IP, 20% of IP loaded) from HeLa cells.  Antibodies: Affinity purified rabbit anti-Hakai antibody A302-968A used for IP at 3 ug/mg lysate.  Hakai was also immunoprecipitated by rabbit anti-Hakai antibody A302-969A, which recognizes a downstream epitope.  For blotting immunoprecipitated Hakai, A302-969A was used at 1 ug/ml.  Detection: Chemiluminescence with an exposure time of 10 seconds.