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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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The cell adhesion protein, GlialCAM, protects MLC1 against ubiquitination and degradation at the ER. (A) The correlation of the PM expression between endogenous GlialCAM (GCAM) and exogenous MLC1-wt was measured using cs-ELISA in HeLa cells and expressed as a percentage of mock-treated MLC1-wt cells. The GCAM downregulation efficiency was altered by using different amounts of siGCAM. (B) Western blot analysis of GCAM downregulation on the cellular expression of MLC1 variants was determined after depleting ~ 60% of GCAM with short hairpin (sh)RNA or shNT (non-targeted). Total MLC1 expression was quantified against the loading control Na⁺–K⁺-ATPase (Na⁺K⁺). (C) GlialCAM overexpression protects MLC1-C326R against rapid ER elimination. Immunoblot analysis of MLC1 and GCAM expression levels was carried out after 20 h BFA (5 µg/ml) or mock-treated HeLa cells with or without GlialCAM overexpression. (D) The ER-confined MLC1-wt and C326R degradation were monitored in fully GCAM (sh/si) depleted or control (NT) HeLa cells. The ER-to-Golgi transport was inhibited with BFA (5 µg/ml) for 20 h, and the ER was cleared using CHX-chase for 30 min with or without proteasomal inhibition (Bortezomib, 1 µM). The absence of complex glycosylation in the hERG channel confirmed BFA efficacy by disrupting ER-to-Golgi transport. (E) The influence of GCAM expression on the PM amount of MLC1-wt was measured using cs-ELISA. Proteasomal inhibition (Bortezomib, 2 h) was unable to rescue the PM expression of MLC1 when GCAM was partially depleted as in panel Fig. 4B. (F) GCAM overexpression effect on the MLC1 ubiquitination at the ER was measured in HeLa cells treated with BFA for 20 h and +/− GCAM. MLC1-wt or C326R were IPed with anti-HA under denaturing conditions. The ubiquitin signal was normalized to MLC1 expression in the IP (left panel). GCAM overexpression decreases the normalized MLC1-C326R ubiquitination by ~ 5-fold (right panel). (G) The overexpression effect of GCAM on MLC1-wt ubiquitination at the ER was measured in cells treated with BFA for 20 h. GCAM was depleted (si/sh) or cells treated with non-target (NT) and proteasomal inhibitor (Bortezomib, 2 h). MLC1-wt was immunoprecipitated with anti-HA under denaturing conditions to measure direct ubiquitination. Means ± SEM, n ≥ 3. ****p < 0.0001.