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. 2010 Dec 15;21(24):4312–4324. doi: 10.1091/mbc.E10-04-0310

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© 2010 by The American Society for Cell Biology

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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Figure 7. — Only unincorporated PSMB5 precursors associate with RhoS. (A) Schematic models showing the structures and autocleavage of different constructs used in this work. Pro, the propeptide in the precursor proteins. (B) Glycerol gradient analysis of the association between RhoS and PSMB5 in the biogenesis of proteasomes. Extracts were prepared from HEK293T cells cotransfected with Flag-Rhos and PSMB5-Myc. Two milligrams of cell lysate were then resolved on a linear glycerol gradient (10–40%), and the fractions were analyzed by Western blot using anti-Flag, anti-Myc, and anti-PSMA2 antibodies. P, precursor; M, matured. (C) Comparing the composition of the protein complexes associated with the RhoS, PSMB5 precursors alone, or both precursor and matured PSMB5 to define which type of PSMB5 associates RhoS. The proteins complex were immunoprecipitated from the indicated cells using anti-Flag antibody and resolved for detecting the existence of RhoS, PSMB5 precursor, PSMA2, and POMP individually. (D) Partial colocalization of RhoS and PSMB5 in the cells. MCF-7 cells coexpressing Flag-RhoS and PSMB5-Myc were fixed and probed with anti-Flag and anti-Myc antibodies. Nuclei were stained with DAPI. Bar, 19.7 μm.