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. 2010 Aug 23;190(4):637–650. doi: 10.1083/jcb.200908092

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© 2010 Minami et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — BAG-6 is essential for CL1 degron-dependent proteasomal degradation. (A) Schematic representation of the 3xFlag-tagged EGFP protein fused with CL1 degron used in this study. (B) CL1 degron-associated proteins identified in this study. After transfection of a 3xFlag-tagged EGFP-CL1 expression vector, HeLa cells were treated with 5 µM MG132 for 4.5 h. Proteins immunoprecipitated with antibody against Flag were subjected to SDS-PAGE and PMF analysis. 3xFlag-tagged EGFP immunoprecipitates were used as a negative control. (C) Knockdown of BAG-6 suppressed the degradation of the CL1 degron substrate. 3xFlag-tagged EGFP-CL1 was expressed in HeLa cells with two distinct shRNA vectors for BAG-6 (siRNA-1 and siRNA-2) or control siRNA. After 60 h of shRNA treatment, whole-cell extracts were prepared and subjected to immunoblot analysis with antibodies against Flag, BAG-6, and actin. (D) Expression patterns of endogenous BAG-6 protein in various adult mouse tissues (top). The anti-Hsp70/Hsc70 blot confirmed equal protein loading (bottom). (E) MG132 treatment stimulated the formation of a larger BAG-6 complex. Extracts of NIH3T3 cells were subjected to gel filtration with Superose 6, and the fractions were subjected to Western blotting with specific antibodies against BAG-6. Cells were cultured with (+) or without (−) 20 µM MG132 before harvesting. (F and G) BAG-6 was associated with the 26S proteasome in HeLa cells. (F) An antibody against BAG-6 was used to immunoprecipitate endogenous BAG-6 protein from extracts of HeLa cells cultured with (+) or without (−) 20 µM MG132 before harvesting. The precipitates were immunoblotted with antibodies to the 19S complex (Rpt5 subunit), 20S proteasome (α5 subunit), and BAG-6. (G) Using an antibody against the 26S proteasome subunit Rpt6, endogenous 26S proteasomes were immunoprecipitated from HeLa cell extracts. HeLa cells were treated with 20 µM MG132 for indicated periods. Immunoglobulin derived from nonimmune mouse serum was used as a negative control. The precipitates were immunoblotted with antibodies against BAG-6, 20S, and 19S complex (Rpt6 subunit).