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. 2018 Oct 2;8:14675. doi: 10.1038/s41598-018-32821-4

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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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The Arc binding site is located in the PICK1 BAR domain. (a) A schematic representation of full length PICK1 and PICK1B highlights the structural domains of the protein (PDZ in green; BAR domain in red; and an acidic region in purple). PICK1B consists of the first 278 amino acids and the last 86 amino acids at the C-terminal end resulting in a truncated protein that is 52 amino acids shorter than the original sequence. (b) HEK293 cells were transfected with PICK1 (lane 1–3, positive control) or PICK1B (lanes 4–6). The lysates were loaded (lane 1 and 4, full lysate as a second positive control) or were subjected to a pull-down with purified GST-Arc (lane 3 and 6) or purified GST alone (lane 2 and 4, negative control), followed by Westernblot (WB) blotted for PICK1. (c–e) Representative intensity images of co-transfected SH-SY5Y cells containing Arc-mCherry and EGFP-PICK1-BAR or (g–i) EGFP-PICK1-PDZ under depolarizing conditions indicate that Arc-mCherry can associate with the PICK1-BAR domain but not the PICK1-PDZ. (f,j) Cross-correlation of the Arc-mCherry and EGFP-PICK1 fluctuation signals during RICS analysis produces (f) a characteristic autocorrelation function (ACF) indicating protein-protein interaction for cells transfected with the PICK1-BAR construct (j) while the PICK1-PDZ domain showed little to no correlation with Arc-mCherry. n = 15 cells for each condition. (The red to light blue distribution indicates the intensity of pixels that have correlated diffusion in both channels. Broader histograms (more red) indicate less correlation of the signals).