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. 2024 Feb 27;10:24. doi: 10.1038/s41421-023-00622-3

Fig. 3. PGAM5 is identified to interact with MARCH2.

Fig. 3

a Comparison of cell type distributions among different groups showing changes in MP and cardiomyocyte clusters after I/R procedure. b, c KEGG enrichment analysis (b) and GO (c) analysis of scRNA-seq data from WT-I/R and MARCH2–/–-I/R hearts. d, e Interaction between NLRP3 and ASC in mitochondria (mito-NLRP3 and mito-ASC) of cardiac tissues of MARCH2 KO and WT mice subjected to I/R was examined by IP-western blotting assay. IP with NLRP3 antibody (d) and IP with ASC antibody (e). f Representative Western blotting analyses of MARCH2, NLRP3, ASC, caspase-1 (procaspase1; cleaved caspase-1), GSDMD (full-length; N-terminal) in cardiac tissues of MARCH2 KO and WT mice subjected to I/R (45 min/9 h). g IL-18 release was measured by enzyme-linked immunosorbent assay (ELISA) in cardiac tissues of MARCH2 KO and WT mice subjected to I/R (45 min/9 h). h Schematic diagram showing MS analysis workflow for identifying targets of MARCH2. i IP analysis with anti-HA antibody and immunoblotting with antibodies of anti-Flag and anti-HA, respectively, in NMCMs transfected with PGAM5-HA or control vector along with MARCH2-Flag. j Endogenous IP of MARCH2 and PGAM5 in NMCMs. k Direct interaction between GST-PGAM5 and His-MARCH2 demonstrated by GST pull-down assays. Both input and pull-down samples were immunoblotted with anti-GST and anti-His antibodies. GST-PGAM5 and His-MARCH2 proteins were expressed in vitro. l Representative immunofluorescence images of MARCH2 and PGAM5 in NMCMs. Data are shown as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.