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. 2023 Jan 23;25(3):467–480. doi: 10.1038/s41556-022-01074-9

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© The Author(s) 2023

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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Fig. 5 — a, Volcano plot of the interactome of HSPB1^S135F versus eGFP obtained from WCL analysis. Mitochondrial proteins are highlighted in red. A one-sided t-test was performed for pairwise comparison of both conditions. Significantly enriched proteins are indicated by curved line, set by an FDR value of 0.05 and S₀ value of 1. b, Co-immunoprecipitation with anti-V5 beads to validate the mitochondrial interactors in WCL. c, Co-immunoprecipitation with anti-V5 beads to validate the mitochondrial interactors in mitochondria that were pre-treated with proteinase K (10 μg ml⁻¹) to remove all non-imported proteins. d, Co-immunoprecipitation with anti-V5 beads to validate the interaction between V5-tagged HSPB1 wild-type (WT), HSPB1 mutants R127W (RW), S135F (SF) or P182L (PL) and SLC25A12. GFP-V5 was used as negative control. e, Determination of the binding sites of HSPB1 on SLC25A12. Immunoprecipitation of SLC25A12 using anti-FLAG beads to pull down SLC25A12-3xFLAG (full-length (FL), N-terminal domain (NTD) or C-terminal domain (CTD)) and assessed for the co-immunoprecipitation of HSPB1^S135F. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-FLAG (SLC25A12), anti-V5 (HSPB1^S135F) and anti-tubulin (loading control) antibodies. Schematic representation of SLC25A12 with FL, NTD or CTD. The transmembrane domain is depicted in grey. f, HEK293 Flp-In cells were depleted from Tafazzin (TAZ) as previously described⁷⁵. cl., clone. Mitochondria were isolated and compared with the cytosolic or the NP-40-soluble WCL fraction. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-HSPB1, anti-Tubulin (cytosolic marker) or anti-VDAC1 (mitochondrial marker) antibodies. Results are representative of two (d) or three replicates (b, c, e and f). Source numerical data and unprocessed blots are available in source data.

Source data