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

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© The Author(s) 2023, corrected publication 2024

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Fig. 4 — a Western blotting analysis of PGAM5-HA and MARCH2-Flag. HL-1 cardiomyocytes were transfected with vector expressing PGAM5-HA or MARCH2-Flag and treated with MCM + H/R. b Western blotting analysis of MARCH2 and PGAM5 proteins in HL-1 cardiomyocytes transfected with MARCH2 siRNA, siRNA-resistant MARCH2 or negative control and treated with MCM + H/R. c Analysis of PGAM5-HA protein levels in MCM + H/R-treated HL-1 cardiomyocytes transfected with either MARCH2 WT or its catalytically inactive mutant (C64/67 S, abbreviated CS). d, e Western blotting analysis of MARCH2 and PGAM5 protein levels and quantification analysis of PGAM5 level. HL-1 cardiomyocytes were transfected with MARCH2 (d) or siRNA-MARCH2 (e) and then treated with MCM + H/R and cycloheximide (CHX; 30 μm). f, g Corresponding quantitation graphs of relative PGAM5 degradation in MCM + H/R-treated HL-1 cardiomyocytes transfected with MARCH2 (f) or siRNA-MARCH2 (g) and CHX. The experiments were repeated three times. Error bars represent standard deviation. h Effects of the indicated polyUb on MARCH2-mediated PGAM5 ubiquitination. HL-1 cardiomyocytes were transfected with the indicated ubiquitin under MCM + H/R treatment. IP analysis with anti-HA antibody and immunoblotting with antibodies of anti-His and anti-HA. i Western blotting of the mapping analysis showing the binding domains of MARCH2 to PGAM5. Various truncated forms of MARCH2 (△1–55, △56–116, △117–214 and △215–246) were expressed and purified. j–l Effects of the indicated PGAM5 KR mutants (K88R, K141R, or combined K88-141R) on MARCH2-mediated PGAM5 ubiquitination (j), protein level (k), and degradation (l) under MCM + H/R treatment. PGAM5^–/– HL-1 cardiomyocytes were transfected with the indicated constructs, and PGAM5 protein levels were analyzed. Statistical differences were analyzed by unpaired Student’s t-test for comparison between two groups. *P < 0.05, **P < 0.01.