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. 2011 Jul 29;6(7):e22979. doi: 10.1371/journal.pone.0022979

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This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.

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(A) Structure of RPN3 and schematic representation of the truncated mutant of RPN3. The position of PCI/PINT domain is as marked. N-terminal truncated RPN3 (residues 128–534 a.a., termed as RPN3[ΔN]) was cloned into pCMV-Myc vector to express a Myc epitope-tagged product. (B) Interaction of TSPX and RPN3 in mammalian cells. Myc-RPN3[ΔN] was co-transfected with p3×FLAG-CMV7 (FLAG control), FLAG-TSPX[ΔPro], FLAG-TSPX[Tail-L] or FLAG-TSPX[ΔProΔC] into 293T cells. Immunoprecipitations were carried out as before using anti-FLAG antibody. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. The immunoblots indicate that human RPN3 co-immunoprecipitates with human TSPX. (C) RPN3 did not interfere the interaction between TSPX and HBx. HA-HBx and FLAG-TSPX[ΔPro] expression vectors were co-transfected with or without Myc-RPN3[ΔN] vector into 293T cells. Immunoprecipitations were carried out using anti-FLAG antibody. Immunoblots were probed with anti-HA, anti-Myc and anti-FLAG antibodies, respectively. The results indicate that interaction between HA-HBx and FLAG-TSPX was not affected by co-expression of Myc-RPN3[ΔN]. (D) Over-expression of RPN3 protected HBx from protein-degradation, and TSPX overcomes the protective effect of RPN3. 293T cells were co-transfected with HA-HBx (0.2 µg/well), Myc-RPN[ΔN] (0.05, 0.1 µg/well), RPN3[full] (0.05, 0.1 µg/well) and/or FLAG-TSPX[ΔPro] (0.05 µg/well) as indicated in the figure. pcDNA-DsRed-V5 expression vector (0.1 µg/well) was co-transfected as the internal control for monitoring the transfection efficiency. Cells were lysed 48 h after transfection, and analyzed by Western blot using indicated antibodies. Although co-transfection of RPN3[full] or RPN3[ΔN] resulted in the increase of HA-HBx, TSPX significantly decreased the level of HBx even in the presence of RPN3. (E) Over-expression of Myc-RPN3[ΔN] did not affect on the transcription of HA-HBx. 293T cells were co-transfected with HA-HBx (0.2 µg/well), Myc-RPN[ΔN] (0.1 µg/well), and/or FLAG-TSPX[ΔPro] (0.05 µg/well) as indicated in figure. pcDNA-DsRed-V5 (0.1 µg/well) was co-transfected as the internal control for monitoring the transfection efficiency. Twenty-four hours after transfection, cells were treated with either vehicle (DMSO) or 25 µM MG132 for additional 24 h. Cells were lysed and analyzed by Western blot using anti-HA, anti-V5, anti-Myc, and anti-FLAG antibodies. Co-transfection of Myc-RPN3[ΔN] increased HA-HBx (DMSO). No significant difference was observed in HA-HBx level in the cells treated with MG132 (+MG132).