Journal: iScience

Article Title: The Drosophila connectome reveals axo-axonic synapses on descending neurons

doi: 10.1016/j.isci.2026.115624

Figure Lengend Snippet: Axcs make cholinergic axo-axonic synapses on the GFs as predicted by the MANC connectome (A) AN08B098-type neurons (green) form axo-axonic connections at distinct locations along giant fiber (red, GF) axons. The scale bars shown are 20 μm. (B) The AN08B098-GF connections reconstructed in Neuroglancer (Neuroglancer scale 530.45 μm/vh) mirror the morphology seen with fluorescence in A . (C) We validated AN08B098-to-GF connectivity using anti-bruchpilot to stain for T-bars in active zones (white) along AN08B098-type neurons and colocalized the resulting fluorescence with the GFs (red), which were filled with tetramethylrhodamine. The scale bars shown are 20 μm. (D) Neuroglancer reveals presynaptic sites at similar locations seen in colocalized fluorescent image (Neuroglancer scale 530.45 μm/vh). (E and F) XY and XZ plane views of the preparation shown in (A) and (B). The zoomed-in 6 μm (scale bars 3 μm) inlays show AN08B098 forming a single synapse with the GF in (E). The yellow arrows detail the precise location AN08B098 forms the Brp-positive chemical synapse (white) to the GFs in (F). Scale bars shown are 5 μm. (G–O) EM images showing monosynaptic connections between single GF (green) and AN08B098 neurons identified by the following MANC id: (G and H) 21041, (I) 21589, (J) 23949, (K) 152261, (L) 16900, (M) 20444, (N) 22275, and (O) 24038. Pre-and postsynaptic sites are detected in EM slices using a 3D convolutional neural network to identify T-bars (cyan dots) and postsynaptic densities (PSDs, magenta dots). (P–S) We expressed anti-choline acetyltransferase (anti-ChAT) and anti-GFP in AN08B098 neurons. Anti-ChAT colocalizes to AN08B098 cells, with particularly bright staining in the cell bodies. This finding suggests acetylcholine synthesis is present within these cells, validating connectome transmitter predictions for AN08B098. Scale bars shown are (P) 20 μm and (Q–S) 5 μm.

Article Snippet: A mouse primary antibody against bruchpilot (1:50, DSHB, NC82) was used to label presynaptic chemical active zones and coupled to secondary Goat anti-mouse Alexa Fluor 647 (1:500, 115-605-003).

Techniques: Fluorescence, Staining

Journal: The Journal of General Physiology

Article Title: Beat-locked ATP microdomains in the sinoatrial node map a Ca 2+ -timed energetic hierarchy and regional pacemaker roles

doi: 10.1085/jgp.202513874

Figure Lengend Snippet: Superior SA node myocytes exhibit elevated diastolic ATP and metabolic flux compared with the inferior region. (A) 3D segmented maximum-intensity projection of a whole-mount SA node immunolabeled for CD31 (vasculature, red) and cyto-iATP (myocytes, green). The dashed line denotes the boundary between superior and inferior regions. (B) Image-processing workflow illustrating merged maximum-intensity projections, binary segmentation masks, and extraction of grayscale cyto-iATP signals used for quantitative analysis. (C) Mean cyto-iATP fluorescence intensity per myocyte, grouped by region ( N = 5 mice per region), reporting expression levels of the EGFP-tagged cyto-iATP sensor. (D) Live confocal imaging of cyto-iATP signals showing representative line-scan images and corresponding normalized fluorescence traces (F/F 0 ) from superior and inferior regions. (E and F) Summary quantification of cyto-iATP signal mass rate (E) and estimated diastolic [ATP] i (F). P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.

Article Snippet: For immunolabeling, SA nodes were incubated for 48 h at 4°C with a goat anti-mouse CD31 primary antibody (1:50, AF3628; R&D Systems).

Techniques: Immunolabeling, Extraction, Fluorescence, Expressing, Imaging

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) PCR amplification results after five blind passages. ( B ) TEM observation of CPV L4. Arrows indicate spherical virus particles; scale bar, 50 nm. ( C ) Cytopathic effects of CPV L4 infection in F81 cells (MOI = 1). ( D ) Western blot analysis of VP2 expression in CPV L4-infected F81 cells (MOI = 1). Upper band was observed above the VP2 protein band, which may be caused by post-translational modification of the VP2 protein or incomplete denaturation. GAPDH served as an internal control. ( E ) Immunofluorescence detection of CPV L4-infected F81 cells (MOI = 1); scale bar, 100 µm. ( F ) Growth kinetics of CPV L4 in F81 cells (MOI = 0.1). Infected cells and supernatants were harvested every 12 h for 72 h. Viral genome copies are shown as log10(copies/µL). Data represent three independent experiments (triplicate each) and are presented as mean ± SEM.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Amplification, Virus, Infection, Western Blot, Expressing, Modification, Control, Immunofluorescence

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: Genetic and evolutionary analysis of CPV VP2. ( A ) Maximum-likelihood phylogenetic tree of the CPV VP2 gene constructed using MEGA X with the JTT+G substitution model and 1000 bootstrap replicates. Orange circles indicate isolates CPV L1–L8. ( B ) Heatmap of amino acid similarity among CPV VP2 proteins. ( C ) VP2 amino acid mutation analysis of isolates CPV L1–L8 relative to prototype CPV-2. ( D ) Visual alignment of partial VP2 sequences between CPV L4 and giant panda-derived CPV-2c strains. The orange arrow indicates the full-length VP2 sequence of CPV L4, and blue arrows indicate reported partial VP2 sequences of four panda-infecting CPV-2c strains. Sequences are aligned by VP2 amino acid positions.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Construct, Mutagenesis, Derivative Assay, Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Schematic of nanobody expression construct. Nanobody genes are expressed under the Pgrac promoter, fused with the amyQ signal peptide for secretion via the Sec pathway, and linked to a 6 × His tag for detection and purification. ( B ) SDS-PAGE analysis of five nanobodies in Bacillus subtilis culture supernatants. ( C ) Western blot identification of His tags for five nanobodies. ( D ) Indirect ELISA evaluation of binding reactivity between nanobodies and immunization VP2 protein; results are shown as OD450 values. * p < 0.05, **** p < 0.0001.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Expressing, Construct, Purification, SDS Page, Western Blot, Indirect ELISA, Binding Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Western blot validation of Nb10 recognition of VP2 in CPV L4-infected cells (MOI = 1); Nb10 served as the primary antibody. ( B ) Immunofluorescence detection of Nb10 recognition at the cellular level (MOI = 1); Nb10 served as the primary antibody; nuclei were stained with DAPI (blue). ( C ) Molecular docking model of Nb10 with CPV VP2 showing potential interaction sites. ( D ) Visualization of the Nb10–VP2 complex from different angles.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Western Blot, Biomarker Discovery, Infection, Immunofluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: ( A ) Schematic of Nb10-Fc expression construct. ( B ) Western blot validation of Nb10-Fc expression. Nb10-Fc’ indicates the non-reducing condition result; Nb10-Fc indicates the reducing condition result. Nb10-Fc was detected under both conditions but showed different migration characteristics. ( C ) Indirect ELISA evaluation of Nb10-Fc binding to immunization VP2 protein (OD450). ( D ) Western blot validation of Nb10-Fc recognition of VP2 in CPV L4-infected cells (MOI = 1). Upper band was observed above the VP2 protein band, which may be caused by post-translational modification of the VP2 protein or incomplete denaturation. GAPDH served as an internal control. ( E ) Immunofluorescence detection of Nb10-Fc recognition in CPV L4-infected cells (MOI = 1). Specific green fluorescence was observed in infected cells; nuclei were stained with DAPI (blue). ( F ) Molecular docking model and interface analysis of Nb10-Fc with CPV VP2. ( G ) Visualization of the Nb10-Fc–VP2 complex from different angles. **** p < 0.0001.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques: Expressing, Construct, Western Blot, Biomarker Discovery, Migration, Indirect ELISA, Binding Assay, Infection, Modification, Control, Immunofluorescence, Fluorescence, Staining

Journal: Animals : an Open Access Journal from MDPI

Article Title: Molecular Characterization of Representative CPV-2c Isolates and Establishment of VP2-Targeted Nanobody-Based Immunodetection Tools

doi: 10.3390/ani16091402

Figure Lengend Snippet: Dynamic property analyses of CPV L4 VP2–Nb10 (left) and CPV L4 VP2–Nb10-Fc (right) complexes. ( A ) RMSD curves; ( B ) RMSF curves (green shaded regions indicate interaction interfaces); ( C ) hydrogen bond numbers; ( D ) SASA curves; ( E ) radius of gyration (Rg) curves.

Article Snippet: For VP2 detection, membranes were incubated with a mouse anti-CPV VP2 primary antibody (Bioss, Beijing, China, bsm-49051M) followed by HRP-conjugated goat anti-mouse IgG (H + L) (Beyotime, Shanghai, China, A0216).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: Mapping the O -Man dependent E-cadherin interactome using IP screening. A , Schematic diagrams and structural model of CDH1 EC domains : ( left ) CDH1 is a transmembrane protein with five EC domains that form cis- and trans interactions; ( middle ) TMTC2 mediates O -Man on CDH1 EC B-strands, while TMTC3 mediates glycosylations on G-strands ( O -Man structures were grafted onto an AlphaFold model of EC4 using the GlycoShape tool – the mannoses are depicted as green sticks and translucent surfaces on recipient serine and threonine residues ); ( right ) schematic of the β-strand arrangement of an EC domain, highlighting O -Man sites ( green dots ) on the B- ( red ) and G- ( blue ) strands of EC2-4. B , Schematic diagram of the IP-MS-based interactome screen applied to CDH1 : Cryomilled cells are distributed to a 96-well plate and combined with different extractants; CDH1-associated complexes are affinity enriched from each extract using an antibody coupled magnetic medium and then analyzed by protein MS; the compositions of the enriched macromolecular assemblies will vary according to the stabilizing/destabilizing responses of the protein constituents and a putative interactome is constituted by the combined results. C , Results of the IP screen using 32 extraction conditions : ( upper ) silver-stained SDS-PAGE gel showing CDH1 capture by IP screening; ( lower ) hierarchical clustering of MS data, with log 2 -transformed protein abundance values from Proteome Discoverer displayed by color. Grey shading in the heatmap indicates proteins not detected (ND). Six extractants, highlighted in red, were selected for further quantitative analysis. Selected reagents present in extraction solutions are labeled with colored dots.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Protein-Protein interactions, Extraction, Staining, SDS Page, Transformation Assay, Quantitative Proteomics, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: I-DIRT screen for CDH1. A , Depiction of the approach : BG1 WT cells and BG1 CDH1::HA cells were cultured in both light and heavy isotope-labeling media for label-swapped I-DIRT experiments. The resulting cell powders were combined in 1:1 (w:w) ratios for IP-MS analyses. Specific CDH1 interactors are enriched in one isotope-labeled channel in MS, while non-specific interactors are quantified comparably in both the heavy and light channels. BG1 CDH1::HA cells cultured in heavy-isotope media were designated ‘I-DIRT,’ while BG1 CDH1::HA cells cultured in light-isotope media were designated ‘I-DIRT swap.’ B , Specific interactors identified across six I-DIRT experimental conditions : The interactors are grouped based on how many times they were identified as specific interactors in six extractants. Orange lines represent interactions identified in this study, while gray lines indicate interactions retrieved from the STRING database . C , overlap of the I-DIRT interactor list with two previously published datasets ( , ): the panel on the right lists the 27 common interactors. Proteins are colored by their identification frequency, as in ( B ).

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Cell Culture, Quantitative Proteomics, Protein-Protein interactions, Labeling

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: Bioinformatic analyses of CDH1 interactors. Gene Ontologies (GO) ( A – C ) and Reactome pathways ( D ) enriched among the specific CDH1 interactors. A , enrichment of GO Cellular Components (CC) associated with CDH1 interactors. B , enrichment of GO Biological Processes (BP) associated with CDH1 interactors. C , enrichment of GO Molecular Functions (MF) associated with CDH1 interactors. D , the enriched Reactome pathways is shown on the left . The specific interactors involved in each pathway are detailed on the right . E , Localizations of select CDH1 interactors : proteins with annotated localizations at the cell surface or extracellular matrix are listed. The X-axis of panels ( A – C ) represents the proportion of proteins enriched in each GO pathway.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: O -Man-dependent CDH1 interactome. A , Average log 2 fold change values for CDH1 interactors in TMTC deficient cell lines: colors represent the average log 2 FC values across the six IP conditions, with red indicating increased interaction with CDH1 and blue indicating decreased interaction with CDH1. The circle size indicates the number of IP conditions where the interactor was significantly changed in different TMTC KO cell lines (log 2 FC ≥ 1 or ≤ −1, and p-adj. value ≤ 0.05). B , Selected interactors across different co-enrichment groups, for distinct IP conditions. Symbols indicate statistical significance, with “∗” representing log 2 FC ≥ 1 or ≤ −1 and p-adj. value ≤ 0.05. C , Interactions dependent on O-mannosylation position :: ( upper - panel ) example interactions affected by O -Man on EC domain G-strands; ( middle - panel ) example interactions affected by O -Man on EC domain B- and G-strands together; ( lower - panel ) ANXA1 exhibits increased co-enrichment when O -Man is depleted from EC domain B-strands (see conditions 16 and 20, KO: TMTC2 ). D , cell adhesion ability in cell lines expressing CDH1 with varying O- Man modification statuses. Data are presented as mean ± SEM ( n = 6). Statistical significance is denoted as follows: ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Expressing, Modification

Journal: Molecular & Cellular Proteomics : MCP

Article Title: O -Mannose Glycosylations Influence E-Cadherin Functional Interactions

doi: 10.1016/j.mcpro.2026.101559

Figure Lengend Snippet: Effects of TMTC knock - out on CDH1 and CDH3 abundance and localization. A , Western blot analysis of endogenous CDH3 abundance in BG1 cells with different TMTC KO statuses. B , Flow cytometry analysis of cell surface CDH1 and CDH3 : ( left ) representative histograms comparing fluorescence intensities in BG1 CDH1::HA cells ( green ), BG1 CDH1::HA/KO:TMTC1-4 cells ( pink ), and BG1 KO:CDH1 negative control cells ( grey ); signals normalized to mode; ( right ) Quantification of fold-change in median fluorescence intensity for surface CDH1 and CDH3 in BG1 CDH1::HA cells relative to BG1 CDH1::HA/KO:TMTC1-4 cells (n = 3). C , representative immunofluorescence images showing cellular localization of CDH1 ( green ) and CDH3 ( red ) in control BG1 CDH1::HA cells ( top panels ) and BG1 CDH1::HA/ KO :TMTC1-4 cells ( bottom panels ). Nuclei were counter-stained with DAPI ( blue ). Scale bar = 10 μm. D , Schematic model of the O-Man-dependent CDH1 interactome : some CDH1 interactors are modulated by O- Man, leading to their decreased or increased co-enrichment, based on changes e.g., in their affinity, localization, and/or abundance.

Article Snippet: Anti-CDH3 (MAB861, R&D systems 1:500 in 10% (v/v) FBS in PBS) and Anti-CDH1 primary antibodies (AF648 R&D systems 1:200 in 10% (v/v) FBS in PBS) were added to the cells and incubated for 1 h. The cells were washed 3 times with 100 μl of 10% (v/v) FBS in PBS and incubated with secondary antibody conjugated to fluorophore (Goat anti-Mouse IgG (H; + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 or Donkey anti-Goat IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 at 1ug/ml in 10% (v/v) FBS in PBS).

Techniques: Knock-Out, Western Blot, Flow Cytometry, Fluorescence, Negative Control, Immunofluorescence, Control, Staining