Interaction of polyubiquitin with p62 UBA is necessary for substrate ubiquitination. PC12 cells were transfected with HA-TrkA along with Myc-tagged wild-type (WT) p62, Myc-p62 ΔUBA (a construct lacking the UBA domain), Myc-p62 UBA mutant 1: L398V (no effect on polyubiquitin binding), Myc-p62 UBA mutant 2: F406V (inhibits polyubiquitin binding); or Myc-p62 UBA mutant 4: L417V (inhibits polyubiquitin binding). Complete characterization of the mutants and the effects on binding polyubiquitin has been previously described [9]. Thirty-six hours after transfection the cells were treated with NGF (50 ng/ml) for 15 min, lysed and immunoprecipitated with HA antibody [23], followed by Western blotting for either ubiquitin or TrkA as shown.