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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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The stoichiometry of α1 governs the ratio of canonical to non-canonical proteasomes in pba3Δ cells. (a) Extracts of WT and pba3Δ yeast overexpressing α1 or not and harboring the indicated cysteine substitutions were crosslinked as described in Materials and Methods, followed by separation by non-reducing SDS-PAGE and immunoblotting with antibodies against His (α4) and G6PD (loading control). Crosslinks between adjacent α4 subunits are indicated as α4^α4 and are quantified below (n = 6; error bars = s.e.m.; ns = not significant, p = 0.1754). **Nonspecific cross-reactive bands. Full-length blot is presented in Supplementary Fig. S8. (b) Equal numbers of cells from the indicated yeast strains expressing empty vector (EV) or α1 from a high-copy plasmid were spotted in six-fold serial dilutions on synthetic complete plates lacking or containing MTX and incubated for three days at 30 °C. (c) Extracts of WT and pba3Δ yeast overexpressing α1 or not and harboring the indicated cysteine substitutions were crosslinked as described in Materials and Methods, followed by separation by non-reducing SDS-PAGE and immunoblotting with antibodies against His (α2) and G6PD (loading control). WT cells served as a positive control for crosslinking (lane 3), which was evident as a high-molecular weight species that could be near-fully ablated by treatment of the crosslinked extract with DTT prior to electrophoresis (lane 6).Crosslinks between adjacent α2 and α3 are indicated as α2^α3 and are quantified below (n = 6; error bars = s.e.m.). **Nonspecific cross-reactive bands. Full-length blot is presented in Supplementary Fig. S8.