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. 2019 Feb 14;9:2012. doi: 10.1038/s41598-018-38353-1

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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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Analysis of Tim50 single mutants. (A) Serial dilutions of Tim50 WT and mutant cells were grown for 5 days at the indicated temperature on SCD (-Leu) and SCG plates. (B) Accumulation of the precursor form of Hsp60 in wild type and mutant cells was analyzed by SDS-PAGE and immunoblot with Hsp60 antibodies, as described under Methods. (C) Tim23-Tim50 interaction in the indicated strains was analyzed using pull down assay with Ni-NTA agarose beads essentially as described in Fig. 1C. T, 10% of total material. B, 100% of bound material. As an example, binding of Tim23 to Ni-NTA agarose beads using D278V/R339S double mutant and its corresponding single mutants was quantified as described in Fig. 1C.