Fig. 7: AnkB-220 interacts with mitochondria and the mitochondrial fission machinery in SKM. | Nature Communications

Fig. 7: AnkB-220 interacts with mitochondria and the mitochondrial fission machinery in SKM.

From: Ankyrin-B modulates mitochondrial fission in skeletal muscle and is required for optimal endurance exercise capacity

Fig. 7

a Western blot of total GC lysates and mitochondria fractions probed with antibodies specific to AnkB and markers of subcellular compartments. n = 3 fractionations. b IP-proteomics-MS/MS workflow used to uncover the AnkB interactome in GC and SOL and validation of AnkB pulldown from SKM (n = 3 AnkB-IP or control IgG-IP). Putative AnkB interactors showed at least a two-fold enrichment in AnkB-IgG IP eluates over control-IgG IP with a p < 0.05 determined by a one-tail t-test. Created with elements of BioRender. Voos, K. (2025) https://BioRender.com/iyaxrha. c, Total (left) and mitochondrial (right) AnkB interactome hits identified in GC and SOL IPs. d Protein-protein interaction map of mitochondrial hits shared between AnkB interactomes in GC and SOL. Nodes and edges respectively represent proteins and protein interactions. e Schematic depicting the role of MFF and Drp1 in mitochondrial fission. f Images showing in situ nanocomplexes between AnkB and either βII-spectrin (f”), MFF (f”’), or Drp1 (f””) in FDB fibers detected by a proximity ligation assay (PLA) (representative of n = 3 independent PLA experiments). A PLA using only anti-AnkB antibodies serves as a negative control (f’). Mitochondria are labeled by endogenous GFP expression. Insets show higher magnification of regions demarked by a yellow dotted box. Scale bar, 3 µm. g MFF-GFP and AnkB-HA co-expression in total lysates and complex formation in GFP-IP eluates from control and Drp1 knockdown HEK293T cells. h Quantification of AnkB-HA/MFF-GFP signal in GFP-IP eluates. i AnkB-GFP and mCherry-Drp1 co-expression in total lysates and complex formation in GFP-IP eluates from control and MFF knockdown HEK293T cells. j Quantification of mCherry-Drp1/AnkB-GFP signal in GFP-IP eluates. mCherry-Drp1 and MFF-GFP co-expression in total lysates and complex formation in GFP-IP eluates from control and AnkB knockdown HEK293T under crosslinking (k) or native IP (m) conditions. l, n Quantification of mCherry-Drp1 monomers or oligomers/MFF-GFP signal in GFP-IP eluates. Data in g–n was collected from n = 3 independent transfections and co-IP/group. Graphs show mean ± S.D. Statistical differences were determined by single or multiple two-tailed unpaired t-tests with a significance threshold of p < 0.05.

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