egfp beta actin 88 addgene plasmids  (Addgene inc)

Journal: bioRxiv

Article Title: Anillin-dependent actin assembly at PML NBs protects genome stability

doi: 10.1101/2025.10.26.679380

Figure Lengend Snippet: a , Representative super-resolution Airyscan microscopy images of U-2OS cells treated with 0.4 µM aphidicolin (APH) for 24 h, showing phalloidin-stained F-actin and PML immunofluorescence. Left: maximum intensity projection; right: Zoomed-in single Z-plane. Scale bar 5µm. b, Representative fixed-cell images of U-2OS cells expressing nuclear actin chromobody (NLS-actin-CB) stained with PML antibodies following 24 h of 0.4 µM APH treatment. Scale bar 5µm. c, Representative live-cell spinning disk confocal microscopy images of U-2OS cells co-expressing NLS-actin-CB, PCNA-CB, and miRFP670-tagged PML treated with 0.4 µM APH for 24 h. Time is indicated in hours:minutes relative to the first time point. Scale bar 5µm. d, Representative immunoblot analysis of endogenous PML immunoprecipitates from U-2OS cells treated for 24 h with DMSO, 0.4 µM APH, or APH in combination with 200 µM CK-666. n = 3 biological replicates. e, Quantification of S-phase nuclei positive for nuclear F-actin from experiments in panel ( c ). Cells were transfected with the indicated siRNAs 48 h before imaging and treated with DMSO or APH for 24 h. Data are mean ± s.e.m. n = 3 biological replicates, scoring ≥31 nuclei per replicate. Statistical analysis by one-way ANOVA followed by Tukey’s post hoc test; P < 0.0001 (****). f, Venn diagram showing the number and overlap of proteins significantly enriched at PML proximity (significantly enriched at Flag-APEX2-PML vs. Flag-APEX2) in U-2OS cells following treatments described in panel ( d ). g, Volcano plots showing enrichment of biotinylated proteins from Flag-APEX2-PML U-2OS cells treated with 24 h of 0.4 µM APH (right) compared to DMSO (left). Significantly enriched PML NB components (log₂ fold change > 0.5, -log p-value > 2.3) are highlighted in red (APH) and blue (DMSO). Statistical analysis by Student’s t-test, n = 3 biological replicates. h, Quantification of normalised Anillin (ANLN) intensity from Flag-APEX2-PML U-2OS samples under the conditions described in ( d ). Statistical analysis by one-way ANOVA followed by Tukey’s post hoc test; P < 0.001 (***), P < 0.0001 (****).

Article Snippet: The NLS-GFP-actin chromobody (Chromotek, acg-n, nuclear-actin-CB) and RFP-PCNA chromobody (Chromotek, ccr, PCNA-CB) were purchased with a material transfer agreement from Chromotek.

Techniques: Microscopy, Staining, Immunofluorescence, Expressing, Confocal Microscopy, Western Blot, Transfection, Imaging

Journal: bioRxiv

Article Title: Ninjurin2 Regulates Vascular Smooth Muscle Cell Phenotypic Switching and Vascular Remodeling through Interacting with PDGF Receptor-β

doi: 10.1101/2025.05.27.656052

Figure Lengend Snippet: A, Subcellular co-localization of Ninjurin2 and PDGFR-β in HeLa cells. Cells were co-transfected with GFP-tagged NINJ2 (pEGFP-N1-NINJ2; green) and Flag-tagged PDGFR-β (p3×Flag-CMV-10-PDGFRB; red). At 48 h post-transfection, cells were fixed, immunostained with anti-Flag primary and Alexa Fluor-conjugated secondary antibodies, and imaged by confocal microscopy. Yellow puncta in merged panels (arrows) indicate membrane- and cytoplasm-localized interaction sites. Scale bars: 10 μm. B–C, Co-immunoprecipitation (Co-IP) confirms direct Ninjurin2–PDGFR-β interaction. HEK293T cells were co-transfected with GFP-NINJ2 and Flag-PDGFR-β. B, Lysates precipitated with anti-Flag magnetic beads were immunoblotted (IB) using anti-GFP antibody. C, Reciprocal Co-IP: GFP-Trap magnetic bead pull-down followed by anti-Flag IB. IgG controls confirmed antibody specificity. Input lanes (5% total lysate) validate expression. IgG was used as a control antibody.

Article Snippet: The slices were then combined with anti-GFP primary antibody (19245S; 1:200; CST) at 4 °C overnight, followed by biotin secondary antibody for 30 min. DAB substrate was used to stain the slides and hematoxylin was used for final detection.

Techniques: Transfection, Confocal Microscopy, Membrane, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Expressing, Control

Journal: bioRxiv

Article Title: Ninjurin2 Regulates Vascular Smooth Muscle Cell Phenotypic Switching and Vascular Remodeling through Interacting with PDGF Receptor-β

doi: 10.1101/2025.05.27.656052

Figure Lengend Snippet: A, Domain architecture of Ninjurin2 and truncated constructs. Schematic depicts full-length Ninjurin2 (top) and deletion mutants used for interaction mapping (bottom), including GFP-NINJ2-aa1-62 contained N terminal region and two N-terminal amphipathic helices (AH1, residues 1–31; AH2, residues 32–62), GFP-NINJ2-aa63-142 contained two transmembrane domains (TM1, 63–97; TM2, 98–125) and C terminal region, GFP-NINJ2-TM1 contained transmembrane domain 1(63–97aa), GFP-NINJ2-TM2 contained transmembrane domain 1(98–125aa), GFP-NINJ2-C contained C terminal region(126-142aa). B, Co-IP analysis showing that GFP-NINJ2-aa63-142 which contained two transmembrane domains (TM1 and TM2) and C terminal region are necessary for PDGFR-β binding. HEK293T cells were cotransfected with PDGFR-β and full length or truncted Ninjurin2 expression plasmids (GFP-NINJ2, GFP-NINJ2-aa1-62,GFP-NINJ2-aa63-142) for 48 h, lysed, and used for immunoprecipitation. Anti-GFP was used for immunoprecipitation and anti-Flag was used for Western blotting. C, Further domain mapping indictaed TM1(63–97aa) and TM2(98–125aa) domains of Ninjurin2 were capable of interacting with PDGFR-β. D, Schematic of PDGFR-β domain architecture and the expression plamid architecture of the co-IP experiments. Truncted PDGFR-β contained tyrosine kinase domain domain (GFP-PDGFRB-TyrKc,600-958aa) and full length PDGFR-β were constructed. E, Co-IP analysis showing that the kinase domain (TyrKc) of PDGFRB interacts with Ninjurin2. HEK293T cells were cotransfected with Flag-Ninjurin2 and GFP tagged PDGFR-β or PDGFRB-TyrKc, Anti-Flag was used for immunoprecipitation and anti-GFP was used for Western blotting.

Article Snippet: The slices were then combined with anti-GFP primary antibody (19245S; 1:200; CST) at 4 °C overnight, followed by biotin secondary antibody for 30 min. DAB substrate was used to stain the slides and hematoxylin was used for final detection.

Techniques: Construct, Co-Immunoprecipitation Assay, Binding Assay, Expressing, Immunoprecipitation, Western Blot