ASB7 interacts with DDA3 and promotes ubiquitination of DDA3. (A) Identification of DDA3 as an ASB7-interacting protein. 3×FLAG-ASB7 expressed in HEK293T cells was purified using anti-FLAG antibody and resolved by SDS-PAGE. ASB7-interacting proteins were analyzed by mass spectrometry. Identified peptides of DDA3 are shown. (B) Interaction between 3×HA-DDA3 and 3×FLAG-ASB7. HEK293T cells expressing 3×HA-DDA3 and 3×FLAG-ASB7 (as indicated) were cultured in the presence of the proteasome inhibitor MG132 (10 µM for 6 h), immunoprecipitated (IP) with anti-HA or anti-FLAG antibody, and immunoblotted (IB) with anti-HA or anti-FLAG antibody. (C) Interaction between endogenous DDA3 and 3×FLAG-ASB7. HeLa cells stably expressing 3×FLAG-ASB7 were cultured in the presence of MG132 (10 µM for 6 h), immunoprecipitated with anti-FLAG antibody, and immunoblotted with anti-DDA3 or anti-FLAG antibody. (D) Interaction between endogenous DDA3 and ASB7. HeLa cells were cultured in the presence of MG132 (10 µM for 6 h), immunoprecipitated with anti-DDA3 antibody, and immunoblotted with anti-DDA3 or anti-ASB7 antibody. (E) Accumulation of DDA3 upon exposure to MG132. HeLa cells were cultured in the presence or absence of 10 µM MG132 for 6 h and then Western blotted with antibodies against DDA3. (F) Down-regulation of DDA3 by overexpression of ASB7. HeLa cells stably expressing 3×FLAG-ASB7 were lysed and immunoblotted with anti-DDA3, anti-FLAG, or anti-Hsp90 antibody. (G) Accumulation of endogenous DDA3 by knockdown of ASB7. Two independent siRNAs (#3 and #5) targeting ASB7 were transfected into HeLa or HEK293T cells, which were cultured for 2 d and then Western blotted with anti-DDA3, anti-FLAG, or anti-Hsp90 antibody. (E–G) Hsp90 is shown as a loading control. (H) ASB7-dependent polyubiquitination of exogenous DDA3 in vivo. HEK293T cells were transfected with plasmids encoding 3×FLAG-ASB7, 3×HA-DDA3, and/or His6-tagged ubiquitin. MG132 (10 µM for 6 h) was used to detect polyubiquitination. Cell lysates were subjected to nickel–nitrilotriacetic acid (Ni-NTA) pulldown to purify proteins modified by His6-ubiquitin, followed by IB analysis with anti-HA, FLAG, or His6 antibody. Asterisks denote nonspecific bands. (I) ASB7-dependent polyubiquitination of endogenous DDA3 in vivo. HeLa cells stably expressing 3×FLAG-ASB7 were examined as in H. (J) Reduced polyubiquitination of endogenous DDA3 by knockdown of ASB7. siRNAs (#3 and #5) targeting ASB7 were transfected into HEK293T cells and examined as in I. (K) ASB7-dependent polyubiquitination of DDA3 in vitro. Recombinant ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (UbcH5a), ASB7 complex (ASB7, Cul5, Elongin B, Elongin C, and Rbx2), His6-ubiquitin, and immunopurified 3×HA-DDA3 were mixed in vitro in various combinations in the presence of ATP, incubated at 28°C for 1 h, and subjected to Western blotting with anti-HA antibody. (L) Quantification of polyubiquitinated DDA3. The signals of polyubiquitinated DDA3 shown in J were quantified. Control sample with or without His6-ubiquitin expression was set as 100 and 0, respectively. *, P < 0.02. Data represent the mean ± SD of three independent experiments.
