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. 2017 Dec 21;39(6):1012–1021. doi: 10.1038/aps.2017.169

Figure 4.

Figure 4

HN interacts with membrane-bound Bax or tBid and inhibits the interaction between Bax and tBid in the membrane. (A) Cy5-labelled Bax (C126S) at 0.5, 1, or 2 nmol/L or membrane-bound Cy5-labelled Bax (C126S) was incubated with 1 nmol/L Cy3-labelled HN or Cy3 dye. In the presence of Cy3-labelled HN or Cy3 dye, FD+A and FD denote the fluorescence of Cy3 at 600 nm with or without Cy5-Bax (C126S). In the absence of Cy3-labelled HN or Cy3 dye, FA and F0 denote the fluorescence of the background at 600 nm with or without Cy5-Bax (C126S). (FD+A-FA) and (FD-F0) denote the emission of Cy3-labelled HN or Cy3 dye at 600 nm with or without Cy5-Bax (C126S). The samples in the buffer were labelled with (B), whereas the samples in liposomes were labelled with (L). Error bars indicate the standard errors, n=3. (B) Cy5-labelled cBid (C3S, C15S, C28S, S76C) at 0.25, 0.5, or 1 nmol/L or membrane-bound Cy5-labelled cBid (C3S, C15S, C28S, S76C) was incubated with 1 nmol/L Cy3-labelled HN or Cy3 dye. In the presence of Cy3-labelled HN or Cy3 dye, FD+A and FD denote the fluorescence of Cy3 at 600 nm with or without Cy5-labelled cBid (C3S, C15S, C28S, S76C). In the absence of Cy3-labelled HN or Cy3 dye, FA and F0 denote the fluorescence of the background at 600 nm with or without Cy5-Bax (C126S). (FD+A-FA) and (FD-F0) denote the emission of Cy3-labelled HN or Cy3 dye at 600 nm with or without Cy5-labelled cBid (C3S, C15S, C28S, S76C). The samples in the buffer were labelled with (B), whereas the samples in liposomes were labelled with (L). Error bars indicate the standard errors, n=3. (C) Cy3-labelled Bax (C126S) at 1 nmol/L was incubated with 0.2 or 5 μmol/L HN, 4 nmol/L Cy5-labelled cBid (C3S, C15S, C28S, S76C) and 1 mg/mL liposomes (93% DPPC and 7% cardiolipin). The fluorescence was measure at the Cy3 excitation wavelength of 532 nm and the Cy5 emission wavelength of 670 nm. Error bars indicate the standard errors, n=3. Data analyzed by an unpaired one-tailed t test. (D) Numbers of fluorescent spots (# of Occurrence) in the images of 100 pmol/L Cy3-labelled Bax (C126S) and 500 pmol/L cBid or 2 pmol/L Cy3-labelled cBid (C3S, C15S, C28S, S76C) in DPPC lipid bilayers with cardiolipin (93% DPPC and 7% cardiolipin) with or without incubation with 100 nmol/L HN. Error bars indicate the standard errors, n=6.