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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 6. — Tethering assay to assess the physical interaction of Mitofusin proteins in trans. A, schematic of the differential epitope labeling utilized in the tethering assay. B, mitochondria were isolated from a clonal population of Mfn1^WT-FLAG-expressing Mfn1-null cells (Mfn1^WT Mfn2^+/+), a clonal population of Mfn1^WTeGFP-expressing Mfn1-null cells (Mfn1^WT Mfn2^+/+), a clonal population of Mfn1^F202LFLAG-expressing Mfn1-null cells (Mfn1^F202L Mfn2^+/+), and a clonal population of Mfn1^F202L-eGFP-expressing Mfn1-null cells (Mfn1^F202L Mfn2^+/+) and incubated with BeF₃ in the absence or presence of GDP. Following lysis, immunoprecipitation was performed with α-FLAG magnetic beads. Proteins eluted from the beads were subjected to SDS-PAGE and immunoblotted with α-Mfn1. The arrowhead indicates the eGFP protein eluted from FLAG beads. Total (T) represents 3% of the input, flow-through (FT) represents 3% of the unbound protein, and elution (E) represents 40% of the immunoprecipitated protein. C, the percentage of the indicated Mitofusin-eGFP in the elution compared with the total is shown as the mean +S.D. of three independent experiments. D, the percentage of the indicated Mitofusin-FLAG in the elution compared with the total is shown as the mean + S.D. of three independent experiments.