Fig 1: The mitochondrial interactome of HSPB1 is enriched for transmembrane proteins of the inner mitochondrial membrane.a, Volcano plot of the interactome of HSPB1S135F 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 HSPB1S135F. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-FLAG (SLC25A12), anti-V5 (HSPB1S135F) 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
Fig 2: CMT disease-causing mutations in HSPB1 disturb its mitochondrial function.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 (MTSCOX8a-HSPB1) and anti-TOM20 (mitochondrial marker). Scale bar, 10 µm. e, Submitochondrial localization of wild-type or P182L-mutant MTSCOX8a-HSPB1 was verified as in b. Samples were analysed by SDS–PAGE followed by immunoblotting using anti-FLAG (MTSCOX8a-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
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