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. 2015 Sep 1;128(17):3187–3196. doi: 10.1242/jcs.165209

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© 2015. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 4. — The Rpn13 C-terminal region inhibits binding of SGTA to the proteasome. (Ai–Bii) Parental HEK293T cells (Ai and Aii) or HEK293^Rpn11-HTBH cells that express an exogenous tagged form of Rpn11 (Bi and Bii) were transiently co-transfected with pcDNA5-SGTA-V5 and empty NpFLAG-CMV2 plasmid (lanes 1 and 2) or NpFLAG-CMV2 encoding the indicated variants of Rpn13 (lanes 3–8). Cells were treated as indicated with 10 µM MG132 or DMSO (solvent control) for 16 h, and then total cell lysates (Ai,Bi), or proteasomal fractions isolated under native conditions using streptavidin beads (Aii,Bii) were prepared. The samples were analysed for endogenous (end.) and exogenous [ex(V5).] SGTA and FLAG-Rpn13 variants (FLAG-Rpn13) by western blotting. Proteasomal recovery was confirmed by western blotting for 20S components and PMSD1 (cf. Fig. 3). *, Non-specific, crossreacting, species detected by certain antibodies.