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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 interacts with NBCn1 and is important for NBCn1 membrane localization. (A) Fluorescence images of NBCn1 (magenta) and RACK1 (green) in non-confluent MDCK-II cells. Nuclei are stained with DAPI (blue). (B) Line scan of marked yellow line in A was carried out in ImageJ and captured across the membrane as illustrated. (C) Fluorescence images of NBCn1 (magenta) and RACK1 (green) in confluent MDCK-II cells. Nuclei stained with DAPI (blue). (D) Line scan of marked line in C was carried out in ImageJ and captured across the membrane as illustrated. Scale bar 10 µm. (E,F) Similar data were obtained in MCF-7 and SKBr-3 cells (Fig. S3A,B). Proximity ligase assay (PLA) carried out in MDCK-II cells. Cells were treated with primary antibodies directed against NBCn1 or RACK1 or only NBCn1 as a control. A positive PLA signal appears as magenta dots. F-actin was stained with phalloidin-488 (green). (F) quantification of PLA dots was carried out in ImageJ using the analyze particles application. PLA signal in 10 randomly chosen cells from 10 different image areas were counted, and the average PLA signal per cell is shown in the bar graph. Data are representative of 3 independent experiments and shown as mean with SEM error bars. ****P < 0.0001, Student’s t test. Similar data were obtained in MCF-7 cells (Fig. S3C,D). (G,H) Native co-IP of NBCn1 and RACK in non-confluent (~70% confluency) MDCK-II cells. Cells were lysed and subjected to pull-down with either antibody against NBCn1 (G) or against RACK1 (H), or the appropriate isotype control, followed by Western blotting of the pull-down and input fractions as shown. Representative of 4 independent experiments. (I,J) The surface fraction of NCBn1 in MCF-7 cells was examined by biotinylation after RACK1 knockdown using two different siRNAs. I: representative Western blots. ß-actin was used as a loading control. J: quantifications of RACK1 and NBCn1 protein expression normalized to mock control. **^,***P < 0.01 and 0.001 vs. scrambled (Scr), respectively, ns: non-significant. n = 3.