Journal: Scientific Reports

Article Title: Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

doi: 10.1038/srep44506

Figure Lengend Snippet: ( a ) F-actin (blue) structure with G-actin protomers ( “n” and “n + 2” ) shown in purple. G-actin structure (purple) with subdomains labeled on the structure. ( b ) Actin treadmilling process for the polymerization and depolymerization of actin filaments. ( c ) TEM images of the NMR samples of U- 13 C, 15 N-CFL2 in complex with F-actin. ( d ) SDS-PAGE of CFL2/F-actin co-sedimentation. Samples containing either F-actin (42 kDa), CFL2 (18 kDa), or CFL2 complexed with F-actin were prepared under the conditions replicating the sample preparation for MAS NMR. Following ultracentrifugation, supernatants (s) and pellets (p) were resolved on SDS-gel.

Article Snippet: Tag-less full-length human CFL2 (cloned between NcoI and BamHI sites in pET15b vector (Novagen)) was expressed in Escherichia coli BL21-CodonPlus(DE3) (Agilent Technologies).

Techniques: Labeling, SDS Page, Sedimentation, Sample Prep, SDS-Gel

Journal: Scientific Reports

Article Title: Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

doi: 10.1038/srep44506

Figure Lengend Snippet: ( a ) 2D NCACX (top), CORD (middle), and NCOCX (bottom) spectra. Selected chemical shift assignments and backbone walks are illustrated in the spectra. ( b ) Backbone walk for the stretch of residues D9-N16 obtained from 3D NCACX spectra (green contours) and 3D NCOCX spectra (black contours). ( c ) Human CFL2 sequence with secondary structure of CFL2 determined by TALOS+, where blue arrows represent β-strands, green boxes represent α-helices and yellow box represents 3 10 helix Colored in yellow and black are residues that are assigned and unassigned (not assigned a single atom), respectively. Spectra were processed with 30 degree sinebell and Lorentizan-to-Gaussian apodization. The first contour is set at 5x the noise level.

Article Snippet: Tag-less full-length human CFL2 (cloned between NcoI and BamHI sites in pET15b vector (Novagen)) was expressed in Escherichia coli BL21-CodonPlus(DE3) (Agilent Technologies).

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

doi: 10.1038/srep44506

Figure Lengend Snippet: ( a ) C α chemical shift perturbations between free CFL2 and CFL2/F-actin. Residues present in dREDOR experiments of CFL2/F-actin are shown with blue bars above the plot and labeled in blue. Chemical shifts of free CFL2 were determined by solution NMR experiments. Chemical shifts of CFL2/F-actin complexes were determined by MAS NMR experiments. ( b ) The residues constituting the corresponding chemical shift perturbations are mapped onto the CFL1 structure (PDB 1Q8G). Chemical shift perturbations between 2–4 ppm are shown in yellow and chemical shift perturbations above 4 ppm are shown in red.

Article Snippet: Tag-less full-length human CFL2 (cloned between NcoI and BamHI sites in pET15b vector (Novagen)) was expressed in Escherichia coli BL21-CodonPlus(DE3) (Agilent Technologies).

Techniques: Labeling

Journal: Scientific Reports

Article Title: Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

doi: 10.1038/srep44506

Figure Lengend Snippet: ( a ) dREDOR-CORD spectra for human CFL2 in complex with F-actin. The residues constituting the corresponding intermolecular CFL2/F-actin interfaces were mapped onto the CFL1 structure (PDB 1Q8G) and are shown in purple in ( b ). Spectra were processed with 30 degree sinebell and Lorentizan-to-Gaussian apodization. The first contour is set at 5x the noise level.

Article Snippet: Tag-less full-length human CFL2 (cloned between NcoI and BamHI sites in pET15b vector (Novagen)) was expressed in Escherichia coli BL21-CodonPlus(DE3) (Agilent Technologies).

Techniques:

Journal: Scientific Reports

Article Title: Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

doi: 10.1038/srep44506

Figure Lengend Snippet: Secondary structure elements are shown above (H denotes α-helices, B – β-strands). Residues previously determined uniquely by solution NMR experiments and cryo-EM studies to be involved in G-actin binding are shown on CFL1 sequence in green and blue, respectively. Residues reported to be involved in G-actin binding in both studies are shown in gray. Residues previously determined by cryo-EM experiments to be involved in the F-actin binding are shown in red. Interface residues determined by dREDOR-based methods are colored in purple on CFL2 sequence. On the primary sequence chemical shift perturbations greater than 2 ppm are indicated by blue asterisk and chemical shift perturbations greater than 4 ppm are indicated by a red asterisk.

Article Snippet: Tag-less full-length human CFL2 (cloned between NcoI and BamHI sites in pET15b vector (Novagen)) was expressed in Escherichia coli BL21-CodonPlus(DE3) (Agilent Technologies).

Techniques: Cryo-EM Sample Prep, Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: Structural Analysis of Human Cofilin 2/Filamentous Actin Assemblies: Atomic-Resolution Insights from Magic Angle Spinning NMR Spectroscopy

doi: 10.1038/srep44506

Figure Lengend Snippet: ( a ) Structure of F-actin (cyan) decorated with CFL2 (gray) determined by cryo-EM (PDB 3J0S) . Two adjacent protomers of actin are shown as cartoons. ( b ) CFL2 interface residues S3, G4, V6, I12, K19, V20, R21, T25, I29, V36, L40, S41, T63, T69, T91, E93, S94, K95, K96, L99, V100, A105, A109, M115, I116, A123, I124, K127, T129, V137, T148, L153, V158, V159, L161 and G163 obtained from dREDOR-CORD MAS NMR experiments of CFL2/F-actin are shown in blue. Subdomains of actin protomers ( “n” and “n + 2” ) are colored in teal (SD1 n , SD1 n+2 ), green (SD2), and cyan (SD3, SD4). DNase binding loop (orange), N- and C-termini (yellow) are indicated on the actin structure. ( c ) Zoomed in region of ( b ) of CFL2 interfaces residues obtained from dREDOR-CORD MAS NMR experiments of CFL2/F-actin are shown in blue. ( d ) Chemical shift perturbations, 2–4 ppm (yellow) and above 4 ppm (red), between CFL2/F-actin and free CFL2 mapped onto cofilin structure. ( e ) Interface residues determined from cryo-EM studies mapped onto cofilin structure . Residues M1-V5, K19-R21, S94-D98, K112-S119 and G154-V158 are shown in orange.

Article Snippet: Tag-less full-length human CFL2 (cloned between NcoI and BamHI sites in pET15b vector (Novagen)) was expressed in Escherichia coli BL21-CodonPlus(DE3) (Agilent Technologies).

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis

doi: 10.3390/ijms23169281

Figure Lengend Snippet: Presence of anti-C1 autoantibodies in LN. Reactivity against C1q ( A ), C1r ( B ) and C1s ( C ) from the plasma of patients with LN (LN) at the time of the first sampling, compared with healthy volunteers (HV), measured by ELISA. Reactivity against C1q ( F ), C1r ( G ) and C1s ( H ) from the plasma of patients with LN (samples with the highest levels of anti-C1 antibodies) (LN), compared with healthy volunteers (HV), measured by ELISA. The dotted line represents the positivity cut-off, determined as average ± 3 SD of the signal, obtained from the plasma of 81 healthy donors. N—normal control IgG, purified from healthy donors. Dose dependences of the IgG binding from different plasma samples to immobilized C1r ( D ) and C1s ( E ) were measured by ELISA.

Article Snippet: Bound C1 complex was revealed by a goat anti-human C1s anti-serum (1:500; Quidel, Eurobio Scientific, Les Ulis, France), followed by a rabbit anti-goat IgG-HRP conjugate (1:2000).

Techniques: Clinical Proteomics, Sampling, Enzyme-linked Immunosorbent Assay, Control, Purification, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis

doi: 10.3390/ijms23169281

Figure Lengend Snippet: Dynamics of the levels of anti-C1 autoantibodies over time. Dynamics of the levels of anti−C1q ( A ), anti−C1r ( B ) and anti−C1s autoantibodies ( C ) over time. Only the patients for whom two or more samples were available are included in the figure. The dotted line represents the positivity cut-off, determined as average ± 3 SD of the signal, obtained from the plasma of 81 healthy donors.

Article Snippet: Bound C1 complex was revealed by a goat anti-human C1s anti-serum (1:500; Quidel, Eurobio Scientific, Les Ulis, France), followed by a rabbit anti-goat IgG-HRP conjugate (1:2000).

Techniques: Clinical Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis

doi: 10.3390/ijms23169281

Figure Lengend Snippet: Correlation analysis between anti-C1q, anti−C1r and anti−C1s antibodies and markers of immunological activity in LN. Correlation between anti−C1q antibodies and levels of C3 ( A ), C4 ( B ), anti−dsDNA ( C ) and ANA titers ( D ). Correlation between anti−C1r antibodies and levels of C3 ( E ), C4 ( F ), anti−dsDNA ( G ) and ANA titers ( H ). Correlation between anti−C1s antibodies and levels of C3 ( I ), C4 ( J ), anti−dsDNA ( K ) and ANA titers ( L ).

Article Snippet: Bound C1 complex was revealed by a goat anti-human C1s anti-serum (1:500; Quidel, Eurobio Scientific, Les Ulis, France), followed by a rabbit anti-goat IgG-HRP conjugate (1:2000).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis

doi: 10.3390/ijms23169281

Figure Lengend Snippet: Comparative analysis between levels of anti-C1q, anti-C1r and anti-C1s in the groups with and without histological signs of LN activity and chronicity.

Article Snippet: Bound C1 complex was revealed by a goat anti-human C1s anti-serum (1:500; Quidel, Eurobio Scientific, Les Ulis, France), followed by a rabbit anti-goat IgG-HRP conjugate (1:2000).

Techniques: Activity Assay

Journal: Acta Neuropathologica Communications

Article Title: A postzygotic de novo NCDN mutation identified in a sporadic FTLD patient results in neurochondrin haploinsufficiency and altered FUS granule dynamics

doi: 10.1186/s40478-022-01314-x

Figure Lengend Snippet: Immunostaining of brain tissue from the FTLD-FET patient. a–d Immunohistochemical lesions in the hippocampus and frontal cortex. Ubiquitin immunolabeling displays rounded intra-cytoplasmic inclusions within the dentate gyrus (black arrows, a ) but with no deformation of the nucleus [OM × 400]. Similar intra-cytoplasmic inclusions were observed in the anterior frontal cortex using FUS immunohistochemistry (black arrows, b ), associated to FUS-positive either rounded (black arrow, c ) or elongated granular intracytoplasmic inclusions (red arrow, c ) [OM × 400] or filamentous curvilinear intranuclear inclusions in the dentate gyrus (black arrow, d ) [OM × 400]. e TAF15 immunostaining displaying normal, finely granular nuclear staining (asterisk) contrasting with loss of TAF15 nuclear immunostaining resulting in cytoplasmic aggregates (black arrows) with f , intracortical neuritic accumulations (black arrow). OM: original magnification

Article Snippet: Immunohistochemical studies were carried out using antibodies directed against α-synuclein (diluted 1/75, Eurobio, les Ulis, France), the PHF tau (AT8, 1/20, Innogenetics, Gent, Belgium), ubiquitin (1/100, Agilent, Les Ulys, France), α-internexin (1/75; Life technologies- Invitrogen, Courtaboeuf, Villebon-sur-Yvette, France), TDP43 (1/1000; Proteintech Europ, Manchester, UK), FUS (1/100; Euromedex, Souffelweyersheim, France), and TAF15 (1/200; Ozyme, St Cyr l’école, France).

Techniques: Immunostaining, Immunohistochemical staining, Immunolabeling, Immunohistochemistry, Staining