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. 2021 Sep 16;11:18435. doi: 10.1038/s41598-021-97777-4

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Early endocytic crosstalk of GlialCAM and MLC1 trafficking dynamics. (A) The relative PM density of endogenous GlialCAM (GCAM) was measured using cs-ELISA in indicated cell lines and expressed as a percentage normalized for cellular proteins. (B) GCAM effect to increase the cellular expression of MLC1 variants (wt, P92S, C326R) was determined by immunoblotting (n = 8). (C) The PM stability of MLC1 variants was measured using cs-ELISA in the presence or absence of exogenous GCAM. (D) Exogenous GCAM enhances MLC1 variants [wt, P92S (PS), C326R (CR)] endosomal recycling, which was measured using cs-ELISA as described in “Materials and methods”. (E,F) The PM expression (E) and stability (F) of GCAM-wt and disease-causing GCAM-R92W were determined using cs-ELISA. GCAM-R92W (~ T_1/2 < 1 h) was destabilized when compared to its wt counterpart (~ T_1/2 2 h). (G) The impact of MLC1 variants [wt, P92S (PS), C326R (CR)] expression on the GCAM internalization was measured using cs-ELISA. (H) Comparison of the PM stability between overexpressed (tr) GCAM and endogenous GCAM (en) and upon coexpression of MLC1-C326R was measured using cs-ELISA. Means ± SEM, n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001.