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. 2019 Apr 1;294(20):8001–8014. doi: 10.1074/jbc.RA118.006347

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© 2019 Engelhart and Hoppins.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 7. — Mfn1^F202L has impaired nucleotide-dependent assembly. A, mitochondria were isolated from a clonal population of Mfn1^WT-expressing Mfn1-null cells (Mfn1^WT Mfn2^+/+) and a clonal population of Mfn1^F202L-expressing Mfn1-null cells (Mfn1^F202L Mfn2^+/+). Mitochondria were either not treated or incubated with the specified nucleotide prior to lysis and then subjected to BN-PAGE and immunoblotting with α-FLAG. The arrowhead denotes the predicted dimer state, arrows denote the larger oligomeric species, and * denotes a nonspecific band. B, the percentage of total protein in the smaller oligomeric state (arrowhead) (predicted dimer) and the higher oligomeric states (arrows) for each condition in A is represented in the bar graph as mean + S.D. of three independent experiments. C, mitochondria were isolated from a clonal population of Mfn1^WT-expressing Mfn1/2 double-null cells (Mfn1^WT Mfn2^−/−) and a clonal population of Mfn1^F202L-expressing Mfn1/2 double-null cells (Mfn1^F202L Mfn2^−/−). Mitochondria were either not treated or incubated with the specified nucleotide prior to lysis and then subjected to BN-PAGE and immunoblotting with α-FLAG. The arrowhead denotes the predicted dimer state, arrows denote the larger oligomeric species, and * denotes a nonspecific band. D, the percentage of total protein in the smaller oligomeric state (arrowhead) (predicted dimer) and the higher oligomeric states (arrows) for each condition in C is represented in the bar graph as mean + S.D. of three independent experiments. E, mitochondria were isolated from a clonal population of Mfn2^WT-expressing Mfn2-null cells (Mfn1^+/+ Mfn2^WT) and a clonal population of Mfn2^F223L-expressing Mfn2-null cells (Mfn1^+/+ Mfn2^F223L). Mitochondria were either not treated or incubated with the specified nucleotide prior to lysis and then subjected to BN-PAGE and immunoblotting with α-FLAG. The arrowhead denotes the predicted dimer state, an arrow denotes the larger oligomeric species, and * denotes a nonspecific band. F, the percentage of total protein in the smaller oligomeric state (arrowhead) (predicted dimer) and the higher oligomeric states (arrow) for each condition in E is represented in the bar graph as mean + S.D. of three independent experiments. PNP, GMPPNP.