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. 2015 Mar 10;10(3):e0119602. doi: 10.1371/journal.pone.0119602

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© 2015 Nakanishi et al

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Fig 2 — A, 15% SDS-PAGE analysis. Lane 1, V_oV₁; lane 2, V_o; lane 3 V₁; lane 4, CL₁₂; lane 5, DF. Molecular weights of each subunit are indicated in parentheses. B, 6% AES-PAGE. The mixtures containing the subcomplexes were incubated for 1h at 25°C respectively, prior to analysis. Lane 1, V_oV₁; lane 2, V_o; lane 3, V₁; lane 4, CL₁₂; lane 5, V_o and V₁; lane 6, V₁ and CL₁₂; lane 7, DF and V_o, lane 8; DF. The band of DF complex was not detected on AES-PAGE gel. C, Gel permeation analysis of complexes and subcomplexes. The mixtures containing subcomplexes indicated by the scheme were incubated for 1h at 25°C respectively, followed by analysis. The molecular weights of each complex are, V_oV₁ (659kDa), V_o (268kDa), V₁ (391 kDa), CL₁₂ (131 kDa) and V₁-DF (37 kDa). D, FRET analysis of reconstituted complexes and subcomplexes. Fluorescence of 3 nM V_1-Cy3 (purple and green lines) or 3 nM V₁-DF_-Cy3 (blue and red lines) was recorded (excitation at 532nm) before and after addition of V_o-Cy5 or CL_12-Cy5. The V_o-Cy5 or CL_12-Cy5 was added at the time indicated by the arrow.