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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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GlialCAM increases the ER stability of MLC1. (A) Immunofluorescence co-localization of MLC1 variants with the ER marker calreticulin. MLC1 variants were detected with indirect immunostaining using an anti-HA primary antibody in astrocytic U251N. Notably, the MLC1-wt displays marginal accumulation in the ER. Bar 5 µm. (B) Cellular turnover of MLC1 variants was monitored upon translational inhibition with cycloheximide (CHX) for 1–6 h. Western blot analysis revealed the fast (gray area) ER-clearance and the slow post-ER pools of MLC1-wt during CHX-chase in comparison to only the fast turnover pools of disease associated mutants. (C) Immunostaining of MLC1 variants before and after CHX chase (1–2 h) in the absence or presence of proteasomal inhibition (B) with Bortezomid (1 µM) during the CHX treatment. MLC1 was detected with an anti-HA antibody and ERp57 was used as an ER marker. Bar 5 µm. (D) The GCAM effect on the total cellular turnover of MLC1 variants was measured by immunoblotting during CHX-chase in HeLa cells. GCAM was overexpressed or depleted ~ 60% with a short hairpin (sh). Non-target shRNA (shNT) was as a control. Non-tr; non-transfected, lys; lysate. Means ± SEM, n ≥ 3.