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. 2018 Oct 23;8:15662. doi: 10.1038/s41598-018-33841-w

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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TMD0 oligomerization. (A,B) To test oligomerization of TMD0 in cellular environment, HEK 293 T cells were transiently transfected with wt (A) and cf variants (B) of TMD0 carrying a C-terminal Flag- or myc-tag. After solubilization with 1% digitonin, co-immunoprecipitation with Flag-tag specific antibody or isotype control (mock) was performed and analyzed by immunoblotting. Input represents 1/25 aliquot of the volume used for immunoprecipitation. Original, uncropped immunoblots are shown in supplementary information. (C) To analyze oligomerization of cf expressed TMD0, precipitates of separately produced cf-TMD0-myc/His and cf-TMD0-Flag/His were mixed, solubilized in LMPG, purified and reconstituted in c6-DHPC. Single or co-purified TMD0 variants (input) were immunoprecipitated by myc-tag specific antibody or isotype control and analyzed by immunoblotting. Input represents 1/15 aliquot of the volume used for pull-down. Original, uncropped immunoblots are shown in supplementary information. (D) To probe the oligomerization dynamics, cf expressed TMD0 variants were separately purified and refolded (input). Cf-TMD0-myc/His and cf-TMD0-Flag/His were mixed in a 1:1 molar ratio and incubated at 4 °C up to five days. Exchange of subunits was analyzed by immunoprecipitation with myc-tag specific antibody. Input represents 1/15 aliquot of the volume used for pull-down. Original, uncropped immunoblots are shown in supplementary information. (E) Oligomerization of MTSL labeled cf-TMD0-His S₂₈C and cf-TMD0-His S₁₃₈C was investigated by PELDOR spectroscopy. PELDOR traces were background corrected (left), and corresponding distance distributions were calculated by Tikhonov regularization (right). (F) Dimer formation of cf-TMD0-His was determined by SEC-MALS. 400 µg of purified cf-TMD0-His were applied on a 15 ml TSK-Gel G3000 SWXL column (green line). Molecular weight distributions were calculated by three-detector method using Astra software and are depicted in black lines (C: protein-detergent conjugate, P: protein, D: detergent).