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. 2021 Mar 15;11:5945. doi: 10.1038/s41598-021-85399-9

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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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Functional validation of novel CARMIL2 variants. (A) Western blot analysis of CARMIL2 expression. Anti-FLAG antibody was used for immunoblotting (left panel). FLAG-TRAF6 served as positive control, β-actin as loading control. The expected molecular weight of CARMIL2 WT and variants (considering the 3xFLAG tag) is shown for comparison (right panel). The black arrow points to the position of the mutation inside the protein. The observed band (~ 180 kDa) of WT CARMIL2 does not correspond to the predicted protein size (~ 155 kDa); this is consistent with what previously reported and with The Human Protein Atlas (available from v19.3.proteinatlas.org)^1,12. Full-length blots are presented in Supplementary Fig. S2. (B) Immunofluorescence staining of HEK293T cells (left panel) and HCT116 cells (right panel) transfected with wild type CARMIL2 or the indicated variants. The first three columns represent the immunofluorescence images of FLAG tagged CARMIL2 (red), actin (green) and nuclei (light blue). The fourth column is a composite image. In HEK293T cells (left panel) CARMIL2 WT and variant p.C155VfsTer54 display a diffuse cytoplasmic expression pattern, while variants p.E978* and p.D623E exhibit a more granular pattern of expression. In HCT116 cells (right panel) expression pattern of all CARMIL2 isoforms is cytoplasmic, although variant p.D623E signal appeared as puncta structures. Scale bar: 7 µm.