Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Fig. 6 — a, Mitochondria were isolated from HeLa cells overexpressing HSPB1 wild-type (WT), HSPB1 mutants R127W (RW), S135F (SF) or P182L (PL) and compared to the cytosol and the NP-40 soluble WCL. Note that the reduced expression of P182L in the WCL is due to reduced solubility in the NP-40-containing buffer. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-V5 (HSPB1), anti-tubulin (cytosolic marker) and anti-VDAC1 (mitochondrial marker) antibodies. The graph represents the densitometric analysis of the mitochondrial fraction after correction for loading using VDAC1 (mean ± s.d.) (n = 3 biologically independent experiments). One-way ANOVA with Dunnett’s multiple comparison test was performed. *P < 0.05, **P < 0.01. NS, non-significant. b, Submitochondrial localization of HSPB1-V5 wild-type (WT) and HSPB1 mutants R127W, S135F and P182L was verified by subjecting intact mitochondria and mitoplasts (derived after osmotic swelling) to proteinase K (PK, 10 μg ml−1) treatment. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-V5 (HSPB1), anti-TOM20 (OM), AIFM1 (IMS), HCCS (IM) and SDHA (matrix) antibodies. c, The same samples as in b were analysed with an anti-HSPB1 antibody to detect both endogenous and exogenous HSPB1. d, A fusion protein between HSPB1 and the MTS of COX8A was expressed in HeLa cells. Immunostaining with anti-FLAG (MTS_COX8a-HSPB1) and anti-TOM20 (mitochondrial marker). Scale bar, 10 μm. e, Submitochondrial localization of wild-type or P182L-mutant MTS_COX8a-HSPB1 was verified as in b. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-FLAG (MTS_COX8a-HSPB1), anti-TOM20 (OM), anti-AIFM1 (IMS), anti-HCCS (IM) and anti-SDHA (matrix) antibodies. Precursor (p) and mature (m) proteins were separated by size as the N-terminal MTS was cleaved off after mitochondrial import. f, Proteinase K protection assay of mitochondria isolated from HeLa cells expressing full-length HSPB1 or C-terminally truncated HSPB1 (delta-CTD). Samples were analysed by SDS–PAGE followed by immunoblotting using anti-FLAG (HSPB1), anti-TOM20 (OM), SLC25A12 (IM) and SDHA (matrix) antibodies. The IPV motif is a conserved C-terminal motif, disrupted by the P182L mutation, and further described in ref. ⁸¹. Results are representative of three replicates (a–f). Source numerical data, including exact P values, and unprocessed blots are available in source data.

Source data