Journal: Human Molecular Genetics

Article Title: Functional characterisation of obesity-associated MRAP2 variants on MC4R and GHSR signalling

doi: 10.1093/hmg/ddag010

Figure Lengend Snippet: Effects of WT or variant MRAP2 on α-MSH-induced β-arrestin-2 (ARRB2) recruitment to MC4R. A-F, dose response curves for MC4R in the absence (control), presence of WT and variant MRAP2 to α-MSH, respectively (expressed as %Emax of control). Values represent mean ± SEM of 3 to 4 independent experiments performed in duplicate. Statistical analyses were performed using one-way ANOVA in which each variant was compared separately with control and WT MRAP2 ( a, P < 0.05 control versus MRAP2; b, P < 0.05 WT MRAP2 versus MRAP2 variants).

Article Snippet: For β-arrestin-2 recruitment NanoBiT-complementation assays MC4R was C-terminally tagged with LgBiT using pBiT2.1-C[TK/LgBiT] (Promega, USA) and co-transfected with human ARRB2 (β-arrestin-2; SinoBiological Europe GmbH, Germany) N-terminally tagged with SmBiT using the pBiT2.1-N[TK/SmBiT] expression plasmid (Promega, USA).

Techniques: Variant Assay, Control

Journal: Human Molecular Genetics

Article Title: Functional characterisation of obesity-associated MRAP2 variants on MC4R and GHSR signalling

doi: 10.1093/hmg/ddag010

Figure Lengend Snippet: Effects of WT or variant MRAP2 on ghrelin-induced β-arrestin-2 (ARRB2) recruitment to GHSR. A-F, dose response curves for GHSR in the absence (control), presence of WT and variant MRAP2 to ghrelin, respectively (expressed as %Emax of control). Values represent mean ± SEM of 3 to 4 independent experiments performed in duplicate. Statistical analyses were performed using one-way ANOVA in which each variant was compared separately with control and WT MRAP2 ( a, P < 0.05 control versus MRAP2; b, P < 0.05 WT MRAP2 versus MRAP2 variants).

Article Snippet: For β-arrestin-2 recruitment NanoBiT-complementation assays MC4R was C-terminally tagged with LgBiT using pBiT2.1-C[TK/LgBiT] (Promega, USA) and co-transfected with human ARRB2 (β-arrestin-2; SinoBiological Europe GmbH, Germany) N-terminally tagged with SmBiT using the pBiT2.1-N[TK/SmBiT] expression plasmid (Promega, USA).

Techniques: Variant Assay, Control

Journal: ACS Omega

Article Title: Identification and Functional Analysis of Dual Nuclear Localization Signals on Desmin

doi: 10.1021/acsomega.5c07336

Figure Lengend Snippet: Putative nuclear localization signals (NLSs) in desmin. A. Schematic representation of three distinct domains: α-helical rod, the head, and the tail. The α-helical rod domain is segmented by three linkers (L1, L12, and L2), forming four coils (Coil 1A, 1B, 2A, and 2B). Both the head and rod domains are crucial for the intermediate filament (IF) assembly, , whereas the tail domain appears to play a role in organizing the IF network. Triangles indicate the locations of NLSs (R: Arginine; S: Serine; E: Glutamic Acid; A: Alanine). Adapted with permission from A cytoplasmic escapee: desmin is going nuclear, Kural Mangıt E, Boustanabadimaralan Düz N, Dinçer P. Turkish Journal of Biology 2021; 45(6):711–719. Copyright 2021 TÜBİTAK. B. Approximative localization of the predicted NLSs on desmin (dotted red squares) on the model from AlphaFold Protein Structure Database (RRID: SCR_023662) (AF- P17661 -F1). , Structure predicted using AlphaFold , and obtained from the AlphaFold Protein Structure Database (DeepMind and EMBL-EBI), CC-BY 4.0.

Article Snippet: The plasmid encoding the full-length desmin protein (pCMV3-DES-GFPSpark, HG13865-ACG) was purchased from Sinobiological.

Techniques:

Journal: ACS Omega

Article Title: Identification and Functional Analysis of Dual Nuclear Localization Signals on Desmin

doi: 10.1021/acsomega.5c07336

Figure Lengend Snippet: Schematic representation of the of the plasmids coding for full-length desmin (pCMV3-DES-GFPSpark) and NLS deletion mutants (pCMV3-DES-GFPSpark Δ R 10‑S42 and pCMV3-DES-GFPSpark Δ E 282‑A313 ) fused to GFP. Boxes with vertical lines indicate linker regions.

Article Snippet: The plasmid encoding the full-length desmin protein (pCMV3-DES-GFPSpark, HG13865-ACG) was purchased from Sinobiological.

Techniques:

Journal: ACS Omega

Article Title: Identification and Functional Analysis of Dual Nuclear Localization Signals on Desmin

doi: 10.1021/acsomega.5c07336

Figure Lengend Snippet: Localization of desmin in nuclei of human skeletal myoblasts. (A). Representative micrographs depicting the nuclear localization of desmin. In cells transfected with pCMV3-DES-GFPSpark desmin is prominently localized within the DAPI boundaries, while Δ R 10-S42 and Δ E 282-A313 desmin mutants (pCMV3-DES-GFPSpark Δ R 10‑S42 and pCMV3-DES-GFPSpark Δ E 282‑A313 ) show minimal or no localization. Third column shows 3D surface rendering of the same area. Confocal z-stack images were deconvolved using Huygens Professional, and surface rendering was performed with the Surface Renderer tool. In cells transfected with pCMV3-DES-GFPSpark, nuclear desmin fluorescence is observed independently of cytoplasmic extensions. In contrast, in cells transfected with plasmids encoding mutant sequences, desmin signal appears as a continuation of the cytoplasmic extensions without distinct nuclear localization. All images shown correspond to the same individual cell, presented using different projections or renderings. (B). Evaluation of colocalization in cells transfected with desmin mutants compared to full-length desmin sequence. Number of cells analyzed for pCMV3-DES-GFPSpark = 101; pCMV3-DES-GFPSpark Δ R 10‑S42 = 118; pCMV3-DES-GFPSpark Δ R 10‑S42 = 110. Mann–Whitney test was used to compare the PCC values (****: p < 0.0001). Error bars were presented as mean ± SD.

Article Snippet: The plasmid encoding the full-length desmin protein (pCMV3-DES-GFPSpark, HG13865-ACG) was purchased from Sinobiological.

Techniques: Transfection, Fluorescence, Mutagenesis, Sequencing, MANN-WHITNEY