Detection of Human and Mouse Hakai by Western Blot (h&m) and Immunoprecipitation (h).  Samples:  Whole cell lysate from HeLa (5, 15 and 50 ug for WB; 1 mg for IP, 20% of IP loaded), 293T (T; 50 ug) and mouse NIH3T3 (M; 50 ug) cells.  Antibodies: Affinity purified rabbit anti-Hakai antibody A302-969A used for WB at 0.1 ug/ml (A) and 1 ug/ml (B) and used for IP at 3 ug/mg lysate.  Hakai was also immunoprecipitated by rabbit anti-Hakai antibody A302-968A, which recognizes an upstream epitope.  Detection: Chemiluminescence with exposure times of 30 seconds (A) and 10 seconds (B).