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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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Extended Data Fig. 7 — a, Membrane association of small heat shock proteins was verified with a sodium carbonate assay. This assay allows the separation of integral membrane proteins (retained in the pellet) from peripheral membrane proteins and soluble proteins (extracted into the supernatant). Mitochondria isolated from HeLa cells were resuspended in Na₂CO₃ set to the indicated pH. Samples were analyzed by SDS-PAGE followed by immunoblotting using anti-HSPB1, anti-HSPB5, anti-HSPB8, anti-CytC (IMS), anti-mtHSP60 (matrix), anti-AIFM1 (IMS), anti-TOM20 (OM), anti-HCCS (IM), and anti-TIM23 (IM) antibodies. T = total; P = pellet; S = supernatant. Unprocessed blots are available in source data. b, Flow cytometry gating strategy and TMRE membrane potential in control or sHSP-depleted HeLa cells. As a negative control, the membrane potential was dissipated by CCCP treatment. Related to Fig. 3d: ungated, non-debris, singlets, TMRE-intensity. FSC: forward scatter, SSC: side scatter, A: Area, H: height, W: width. Results are representative of one (b), in which more than 130,000 cells were counted per condition, or three (a) replicates. Unprocessed blots are available in source data.

Source data