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. 2019 Aug 27;9:12418. doi: 10.1038/s41598-019-48889-5

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Proteasome subunit α1 is stoichiometrically limiting for proteasome α-ring assembly. (a) Whole cell lysates of yeast strains expressing the indicated 3xFLAG-tagged α-subunits from their chromosomal loci were separated by SDS-PAGE and immunoblotted with antibodies against FLAG, Rpt1, or G6PD (loading control). Quantification of α-subunit expression levels, normalized to the G6PD loading control, is shown to the right (n = 3; error bars = s.e.m.). Full-length blot is presented in Supplementary Fig. S8. (b) Cell extracts prepared from yeast strains expressing 3xFLAG-tagged α-subunits from their chromosomal loci were separated via blue native PAGE and immunoblotted with antibodies against FLAG. Samples were prepared under conditions favoring dissociation of the RP from CP from the 26S proteasome to directly examine subunit abundance in the context of the CP only. Triangles denote migrations of purified CP (filled) and a 20 kDa standard (open). Quantification of the percentage of free α-subunit (shown to the right) was determined by dividing the band corresponding to free α-subunit by the sum of the free and assembled (CP band) subunit abundance for each lane (n = 4; error bars = s.e.m.). ^, Blm10-CP; **, CP assembly intermediate. Full-length blot is presented in Supplementary Fig. S8. (c) Cell extracts prepared from yeast strains expressing the indicated 3xFLAG-tagged α-subunits from their chromosomal loci were fractionated by gel filtration chromatography. Peak fractions corresponding to free α-subunits and 26S proteasomes were separated via SDS-PAGE followed by immunoblotting with antibodies against FLAG. The percentage of free α-subunit (shown to the right) was determined by dividing the band corresponding to free subunit (free) by the sum of the free and 26S band intensities for each lane. (n = 8; error bars = s.e.m.). Full-length blot is presented in Supplementary Fig. S8.