Prevention of polyubiquitination of DDA3 by MTs. (A) Interaction between ASB7 and nonphosphorylated or phosphorylated DDA3. HeLa cells stably expressing 3×FLAG-ASB7 or control cells were treated with 0.1 µg/ml nocodazole or 1 µM taxol for 16 h. Cells were further incubated in the presence of 10 µM MG132 for 4 h, and suspended cells in the culture medium were lysed, immunoprecipitated (IP) with anti-FLAG antibody, and immunoblotted (IB) with anti-DDA3 or anti-FLAG antibody. As, asynchronized. (B) Prevention of polyubiquitination of DDA3 by MTs in a dose-dependent manner. Different amounts of MTs were added to the in vitro polyubiquitination reactions. (C) MTs had no effect on polyubiquitination of Cit2. Different amounts of MTs were added to the in vitro polyubiquitination reactions. (D) ASB7 does not interact with MTs. Whole-cell lysates (input) of HeLa cells were prepared and incubated with or without taxol-stabilized MTs. Samples were centrifuged, and supernatant (S) and pellet (P) fractions were analyzed by immunoblotting with antibodies against DDA3, ASB7, Cul5, α-tubulin, or Hsp90. Hsp90 does not bind to MTs and was used as a negative control.