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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 5. — Mitochondrial in vitro fusion assay reveals a fusion defect for Mfn1^F202L and Mfn2^F223L. A, mitochondria were isolated from WT cells (Mfn1^+/+ Mfn2^+/+), a clonal population of Mfn1^WT-expressing Mfn1-null cells (Mfn1^WT Mfn2^+/+), a clonal population of Mfn1^F202L-expressing Mfn1-null cells (Mfn1^F202L Mfn2^+/+), a clonal population of Mfn1-null cells transduced with empty vector (Mfn1^−/− Mfn2^+/+), a clonal population of Mfn2^WT-expressing Mfn2-null cells (Mfn1^+/+ Mfn2^WT), a clonal population of Mfn2^F223L-expressing Mfn2-null cells (Mfn1^+/+ Mfn2^F223L), and a clonal population of Mfn2-null cells transduced with empty vector (Mfn1^+/+ Mfn2^−/−). The indicated mitochondrial combinations were subjected to in vitro fusion conditions at 37 °C for 30 min. B, mitochondria were isolated from WT cells (Mfn1^+/+ Mfn2^+/+), a clonal population of Mfn1^F202L-expressing Mfn1-null cells (Mfn1^F202L Mfn2^+/+), a clonal population of Mfn1-null cells transduced with empty vector (Mfn1^−/− Mfn2^+/+), a clonal population of Mfn2^F223L-expressing Mfn2-null cells (Mfn1^+/+ Mfn2^F223L), and a clonal population of Mfn2-null cells transduced with empty vector (Mfn1^+/+ Mfn2^−/−). The indicated mitochondrial combinations were subjected to in vitro fusion conditions in the presence of cytosol-enriched fraction at 37 °C for 30 min. C, mitochondria were isolated from WT cells (Mfn1^+/+ Mfn2^+/+), a clonal population of Mfn1^F202L-expressing Mfn1-null cells (Mfn1^F202L Mfn2^+/+), a clonal population of Mfn1-null cells transduced with empty vector (Mfn1^−/− Mfn2^+/+), a clonal population of Mfn2^F223L-expressing Mfn2-null cells (Mfn1^+/+ Mfn2^F223L), and a clonal population of Mfn2-null cells transduced with empty vector (Mfn1^+/+Mfn2^−/−). The indicated mitochondrial combinations were subjected to in vitro fusion conditions at 37 °C for 30 min. D, mitochondria were isolated from WT cells (Mfn1^+/+ Mfn2^+/+), Mfn1-null cells (Mfn1^−/− Mfn2^+/+), Mfn2-null cells (Mfn1^+/+ Mfn2^−/−), 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^−/−). The indicated mitochondrial combinations were subjected to in vitro fusion conditions at 37 °C for 60 min. Data are expressed as a percentage of WT control reactions performed in parallel. Error bars indicate mean + S.D. from at least four independent experiments, and paired Student's t test analysis was performed to determine statistical significance *, p < 0.05; n.s., not significant.