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. 2018 May 9;8:7435. doi: 10.1038/s41598-018-25059-7

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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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RACK1 interaction with FLAG-NBCn1 deletion constructs. (A) Overview of NBCn1 organization and the four FLAG-tagged NBCn1 constructs employed. HEK293 cells were transiently transfected with one full length (137) and three truncated (22, 41 and 122) FLAG-tagged versions of the NBCn1 C-terminal prior to lysis or PFA fixation after 24 h incubation. (B) Fixed cells were prepared for immunofluorescence microscopy using antibodies directed against the most distal C-terminal amino acids in NBCn1 (NBCn1 C-term) or FLAG. Scale bars: 20 µm. (C) Samples from lysed cells were subjected to SDS-PAGE and Western blotting against FLAG and β-actin (loading control). (D) Samples from lysed cells were subjected to FLAG IP and analyzed by SDS-PAGE and Western blotting against RACK1 and p150 (loading control). Ctrl: isotype control. Sham: sham-transfected cells. (E) HEK293 cells were transfected with empty vector pFLAG-CMV-2 (EV) or full length NCBn1 C-terminus (137) and lysed before performing IP against FLAG and analyzing by SDS-PAGE and Western blotting against RACK1 and β-actin (loading control). Representative images from 3–4 independent experiments are shown.