Figure 2.
Figure 2. Destabilization of DDA3 by ASB7. (A) HEK293T cells expressing 3×HA-DDA3 with or without 3×FLAG-ASB7 were exposed to 50 µg/ml CHX for 40, 80, or 120 min. The lysates were subjected to Western blotting with antibodies against HA, FLAG, or Hsp90. (B) The intensities of HA-DDA3 bands in A were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value without ASB7 expression at 0 h. (C) Stabilization of endogenous DDA3 by knockdown of ASB7. Two independent siRNAs (#3 and #5) targeting ASB7 were transfected into HeLa cells and cultured for 2 d, followed by CHX treatment as in A. The lysates were subjected to Western blotting with antibodies against DDA3, ASB7, or Hsp90. (D) The intensities of DDA3 bands in C were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. (E) ASB7 interacts with Cul5 but not Cul2. HEK293T cells expressing 3×FLAG-ASB7 or 3×FLAG-pVHL (Cul2-type ubiquitin ligase as a control) were cultured in the presence of MG132 (10 µM for 6 h), immunoprecipitated (IP) with anti-FLAG antibody, and immunoblotted (IB) with anti-Cul2, anti-Cul5, or anti-FLAG antibody. (F) Schematic representation of ASB7 mutants used in this study. (G) Interaction between the SOCS box of ASB7 and Cul5. HeLa cells stably expressing 3×FLAG-ASB7(WT or ΔSOCS box) were lysed, immunoprecipitated with anti-FLAG antibody, and immunoblotted with anti-Cul2, anti-Cul5, or anti-FLAG antibody. (E and G) Asterisk denotes a nonspecific band. (H) Inactivation of ASB7 by deletion of the SOCS box. HeLa cells stably expressing 3×FLAG-ASB7(WT or ΔSOCS box) were exposed to CHX as in A. The lysates were subjected to Western blotting with antibodies against DDA3, FLAG, or Hsp90. (I) The intensities of DDA3 bands in H were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. (J) Endogenous DDA3 was stabilized by knockdown of Cul5, but not Cul2. Two independent siRNAs (#1 and #2) targeting Cul2 or Cul5 were transfected into HeLa cells and cultured for 2 d, followed by CHX treatment as in A. The lysates were subjected to Western blotting with antibodies against DDA3, Cul2, Cul5, or Hsp90. (A, C, H, and J)_Hsp90 is shown as a loading control. (K) The intensities of DDA3 bands in J were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. For all graphs, *, P < 0.05; **, P < 0.01. Data represent the mean ± SD of three independent experiments (B, D, and I) and four independent experiments (K).

Destabilization of DDA3 by ASB7. (A) HEK293T cells expressing 3×HA-DDA3 with or without 3×FLAG-ASB7 were exposed to 50 µg/ml CHX for 40, 80, or 120 min. The lysates were subjected to Western blotting with antibodies against HA, FLAG, or Hsp90. (B) The intensities of HA-DDA3 bands in A were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value without ASB7 expression at 0 h. (C) Stabilization of endogenous DDA3 by knockdown of ASB7. Two independent siRNAs (#3 and #5) targeting ASB7 were transfected into HeLa cells and cultured for 2 d, followed by CHX treatment as in A. The lysates were subjected to Western blotting with antibodies against DDA3, ASB7, or Hsp90. (D) The intensities of DDA3 bands in C were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. (E) ASB7 interacts with Cul5 but not Cul2. HEK293T cells expressing 3×FLAG-ASB7 or 3×FLAG-pVHL (Cul2-type ubiquitin ligase as a control) were cultured in the presence of MG132 (10 µM for 6 h), immunoprecipitated (IP) with anti-FLAG antibody, and immunoblotted (IB) with anti-Cul2, anti-Cul5, or anti-FLAG antibody. (F) Schematic representation of ASB7 mutants used in this study. (G) Interaction between the SOCS box of ASB7 and Cul5. HeLa cells stably expressing 3×FLAG-ASB7(WT or ΔSOCS box) were lysed, immunoprecipitated with anti-FLAG antibody, and immunoblotted with anti-Cul2, anti-Cul5, or anti-FLAG antibody. (E and G) Asterisk denotes a nonspecific band. (H) Inactivation of ASB7 by deletion of the SOCS box. HeLa cells stably expressing 3×FLAG-ASB7(WT or ΔSOCS box) were exposed to CHX as in A. The lysates were subjected to Western blotting with antibodies against DDA3, FLAG, or Hsp90. (I) The intensities of DDA3 bands in H were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. (J) Endogenous DDA3 was stabilized by knockdown of Cul5, but not Cul2. Two independent siRNAs (#1 and #2) targeting Cul2 or Cul5 were transfected into HeLa cells and cultured for 2 d, followed by CHX treatment as in A. The lysates were subjected to Western blotting with antibodies against DDA3, Cul2, Cul5, or Hsp90. (A, C, H, and J)_Hsp90 is shown as a loading control. (K) The intensities of DDA3 bands in J were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. For all graphs, *, P < 0.05; **, P < 0.01. Data represent the mean ± SD of three independent experiments (B, D, and I) and four independent experiments (K).

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