E CDH13 variants studied here. We also observed similar expression levels and localization of wild type and variant CDH13 in living HEK293 cells expressing GFP-CDH13 fusion proteins (see supplementary information). Tcadherin is a GPI anchored plasma membrane protein. Canonical processing of GPI-anchored proteins involves C-terminal cleavage and attachment of a GPI anchor in the endoplasmic reticulum (ER), followed by Golgi ediated plasma membrane transport and localization [57]. This is schematically presented in Fig. 1. The wild type and all the variant GFP-CDH13 proteins showed comparable expression levels as GFP-CDH13 fusion proteins of approximately 131 kDa, and as C-terminally processed GFPlacking CDH13 proteins of approximately 105 kDa (Fig. S1A and S1B). All the fusion proteins (green) also showed similar distribution in the cytoplasm in unstained living cells (Fig. S2), whereas the C-terminally processed CDH13 (red) showed plasma membrane distribution in fixed and CDH13 stained cells (Fig. S3). Although GFP tags have been used successfully to study Ncadherin [58] and E-cadherin [59] function there is also the possibility that this tag might interfere with the normal processing and functions of the protein being studied. Thus, in addition to plasma membrane localized CDH13 (red signal) (Fig. S3) we also observed a strong green signal from all the wild type and mutant CDH13-GFP fusion proteins (Fig. S2 and S3) in the cytoplasm. However, we consider that this signal, which was not observed in CHO cells expressing the native proteins, is a GFP-related artefactPLOS ONE | www.plosone.organd is therefore not biologically relevant. This illustrates that GFPtags should be used with caution. Despite this limitation, the GFPfusion expression model facilitated the observation of canonical GPI anchor processing of CDH13 with the concomitant expression of CDH13 lacking the GFP tag on the cell membrane. In summary, the cytoplasmic signal from the fusion proteins which can be considered a related GFP artefact does not affect our conclusions that, as the native proteins in CHO cells, the wild type and variant CDH13- GFP fusion proteins were equally expressed, processed and localized on the cell membrane.ConclusionsIn this study we tested the association of CDH13 with adult ADHD by sequencing the CDH13 gene in a Norwegian sample of ADHD patients and controls. This was followed by genotyping the identified CDH13 variants in a larger sample. However, assuming a moderate effect size, this study is probably underpowered to detect significant associations between rare CDH13 variants and ADHD.Fludarabine To investigate the functional effects of CDH13 variants we expressed the wild type protein and the missense variants as native CDH13 in CHO cells and as GFP fusion proteins in HEK293 cells.Tenofovir Disoproxil In both models, we could observe the canonical processing of CDH13 as a GPI anchored protein.PMID:35227773 In the HEK293 cell lines a C-terminal sequence, which also includes a GFP tag, is cleaved off and replaced by a GPI anchor. We obtained similar results from the two over-expression models we used showing comparable levels of protein expression and cell membrane localization of wild type and variant CDH13 proteins. This however, does not exclude the possibility that these CDH13 variants may affect some functions of CDH13 that have not been examined in the current study.Supporting InformationFigure SExpression levels of wild type and variant GFP-CDH13 fusion proteins in HEK293 cells. We.