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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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Isolation and characterization of temperature sensitive mutants of Tim50. (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) Mitochondria isolated from WT cells, or from cells expressing indicated His tagged versions of Tim50 were solubilized with 1% digitonin and incubated with Ni-NTA agarose beads at 4 °C. Following washing steps, specifically bound proteins were eluted with Laemmli buffer containing 300 mM imidazole. Samples were analyzed by SDS-PAGE and immunoblotting with Tim50, Tim17 and Tim23 antibodies. T, UB, 10% of total and unbound material. B, 100% of bound material. Binding of Tim23 was quantified from three independent experiments for His-tagged WT, L194H, A221D/D337V, N283Y/D293N and D2787V/R339S mitochondria and from two for His#1 and His#2 mitochondria. The efficiency of Tim23 binding to Ni-NTA agarose beads with mitochondria isolated from cells expressing His-tagged but otherwise WT version of Tim50 was set to 100%.