Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: iNOS increases sensitivity to cisplatin in ovarian cancer (A) The cell viability of OVCAR8 cells was assessed using RealTime-Glo MT Cell Viability Assay after culturing in increasing concentrations of cisplatin for 72 h. (B) OVCAR8 cells incubated with or without L-NMMA for 48 h, measured by RealTime-Glo MT Cell Viability Assay. (C) Cell viability after either no treatment (control), cisplatin, and a combination of cisplatin and L-NMMA by RealTime-Glo MT Cell Viability Assay. (D) Western blot analysis of vimentin protein expression in ovarian cancer cell lines. (E) Ovarian cancer cell lines were analyzed for VIM mRNA levels by qPCR. (F) OVCAR8 cells were treated with or without L-NMMA (4, 6, 8, and 10 mM) for 48 h, and western blot was used to analyze the effect of L-NMMA on the vimentin protein expression. B-actin was used as a loading control. Band densities were quantified using ImageJ analysis. Error bars, SEM. ∗ p < 0.05; ∗∗ p < 0.01 (compared with the control group, using two-way ANOVA). All experiments were independently repeated three times.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Viability Assay, Incubation, Control, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: iNOS knockout increased chemosensitivity and impaired cell motility and migration of ovarian cancer cells (A) Immunoblotting showing reduced iNOS (left) and vimentin (right) expression levels following NOS2 knockdown in the OVCAR8 cells. (B) NOS2 knockdown sensitized OVCAR8 cells to cisplatin, reducing its IC 50 value. Log-logistic model was used to analyze the data. The group comparison between control group and KO-1 had a p value = 8.98e-07. The comparison between control and KO-2 had a p value = 0.0132. The detected EC50 for control group was 0.8554, for KO-1 was 0.6037, for KO2 was 0.7688. (C and D) Analysis of reduced protein (left) and mRNA (right) expression of iNOS and vimentin following siRNA transfection in OVCAR8 and A2780cis cells for 72 h, assessed by western blot and RT-qPCR. (E) OVCAR8 cells were transfected with 2 different siRNAs or the scramble siRNA and were assessed for migration using the scratch wound assay. The area of the wound was measured at 0, 12, 24, and 36 h by the IncuCyte live-cell analysis system. Two-way repeated measures ANOVA was used to analyze the data. After 6 h, siRNA1 had p value = 1, siRNA2 had p value = 0.23. After 12 h, siRNA1 had p value = 0.71 and siRNA 2 had p value = 0.16. After 24 h, siRNA1 had a p value = 0.39 and siRNA2 had a p value = 0.05. After 36 h, siRNA1 had a p value = 0.86 and siRNA2 had a p value = 0.04. (F) NOS2 KO-1 and KO-2 OVCAR8 cells formed significantly fewer colonies compared to parental OVCAR8. The experiment was performed in triplicate with three biological replicates. Statistical analysis was conducted with two-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01. (G) Silencing of NOS2 by two different siRNAs significantly reduced the number of colonies formed by OVCAR8 cells. Clonogenic growth was measured after 10 days, quantified using ImageJ, and represented as a bar graph (mean ± SEM). The experiment was performed in triplicate with three biological replicates. Statistical analysis was conducted with two-way ANOVA. ∗ p < 0.05 and ∗∗ p < 0.01. All experiments were independently repeated two to three times.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Knock-Out, Migration, Western Blot, Expressing, Knockdown, Comparison, Control, Transfection, Quantitative RT-PCR, Scratch Wound Assay Assay, Cell Analysis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nitric oxide-dependent stabilization of vimentin confers chemoresistance in ovarian cancer

doi: 10.1016/j.omtn.2026.102924

Figure Lengend Snippet: L-NMMA promotes vimentin destabilization by enhancing its ubiquitination (A) CHX chase assay showing vimentin protein levels in OVCAR8 cells treated with 10 mM L-NMMA at different time points. (B) Vimentin ubiquitination was assessed in OVCAR8 cells treated with L-NMMA for 24 h, in the presence of the proteasome inhibitor MG-132 (10 μM) for the final 4 h, followed by immunoprecipitation and western blot using an anti-ubiquitin antibody. (C) Measurement of vimentin S-nitrosylation levels was performed by immunoprecipitation in OVCAR8 cells. (D) Western blot analysis of vimentin expression in OVCAR8 cells treated with 10 mM L-NMMA alone or in combination with MG132 at 1, 5, or 10 μM. (E) Tumor growth and proliferation were monitored in mice bearing parental and NOS2 KO OVCAR8 tumors, evaluated by ROI measurements every 4 days ( n = 6). (F) Kaplan-Meier survival curves of mice bearing parental and NOS2 KO OVCAR8 tumors following cisplatin treatment ( n = 6). Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA for growth curves and the Kaplan-Meier method for survival analysis ( p < 0.05, ∗ p < 0.01). Experiments were performed in duplicate or triplicate.

Article Snippet: The following primers for TaqMan Gene Expression Assay were purchased from Thermo Fisher Scientific; human VIM (Cat. #Hs00958111_m1) and human NOS2 (Cat. #Hs01075529_m1).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 drives tumor-educated M2-like polarization of macrophages and promotes tumor-cell aggressiveness. (A) Workflow for generating TC-hMDMs and TC-BMDMs, circSMAD4 knockdown, and downstream functional assays. (B) RT–qPCR analysis of M1-associated markers (MHC-II [HLA-DRA in TC-hMDMs; H2-Ab1 in TC-BMDMs], NOS2, and CD86) and M2-associated markers (CD163, CD206, and ARG1) in TC-hMDMs and TC-BMDMs. (C) Representative flow-cytometry histograms for HLA-DR, iNOS, CD86, CD163, CD206, and ARG1 in TC-hMDMs. Gating strategy and marker thresholds were defined based on FMO controls (see ). (D) Flow-cytometry quantification of marker-positive cells in TC-hMDMs and TC-BMDMs. (E) ELISA of IL-10, TGF-β, and iNOS in culture supernatants. (F) CCK-8 assays of A549 and LLC cells. (G) Colony-formation assays of A549 and LLC cells with quantification. (H) Bioluminescence-based growth readouts of patient-derived LUAD organoids (PDO #1 and PDO #2) after co-culture with TC-hMDMs. (I) Immunoblot analysis of EMT-related proteins (E-cadherin, N-cadherin, Vimentin) in A549 and LLC cells. (J) Transwell migration and invasion assays of A549 and LLC cells with quantification. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Sections were incubated with primary antibodies against Ki-67 (Servicebio, Cat# GB111499 ), E-cadherin (Proteintech, Cat# 20874-1-AP), and Vimentin (Proteintech, Cat# 10366-1-AP).

Techniques: Knockdown, Functional Assay, Quantitative RT-PCR, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Derivative Assay, Co-Culture Assay, Western Blot, Migration

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 depletion in macrophages restrains LUAD growth and metastasis in vivo. (A) Schematic of orthotopic lung implantation and experimental metastasis models using LLC cells mixed with BMDMs expressing shNC or sh-circSMAD4. (B) Representative images of orthotopic lung tumors. (C) Tumor weight of orthotopic implants. (D) Overall survival of mice bearing orthotopic tumors. (E) Immunofluorescence showing F4/80 and circSMAD4 signals in tumor tissues. Scale bar, 50 μm. (F, G) Representative Ki-67 IHC staining and quantification in orthotopic tumors. Scale bar, 50 μm. (H) Representative bioluminescence images of lung tumor burden in the metastasis model. (I) Tumor weight in the metastasis model. (J) Overall survival of mice in the metastasis model. (K–M) Representative IHC staining and quantification of E-cadherin and vimentin in tumors. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Sections were incubated with primary antibodies against Ki-67 (Servicebio, Cat# GB111499 ), E-cadherin (Proteintech, Cat# 20874-1-AP), and Vimentin (Proteintech, Cat# 10366-1-AP).

Techniques: In Vivo, Expressing, Immunofluorescence, Immunohistochemistry

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: MSC-mt alleviates oxidative stress and promote tissue regeneration during wound healing (A) In vivo imaging showing the spatial–temporal persistence of fluorescently labeled MSC-mt (mtH) at the wound site at indicated time point, indicating transient but sustained early presence after topical application. (B) Measurement of ATP levels in peri-wound tissues on PWD8 showed enhanced local metabolic activity following mtH treatment. n = 5 ∼ 6 per group. (C) Quantification of malondialdehyde (MDA) levels in peri-wound tissues on PWD8 indicated reduced lipid peroxidation and oxidative stress in both MSC-mt–treated wounds. n = 5 ∼ 6 per group. (D) Laser speckle contrast imaging of blood perfusion at the wound site on PWD8 showed improved microvascular perfusion following mtH treatment. n = 5 per group. (E) Representative immunofluorescence images and quantification of CD31 expression in peri-wound tissues on PWD8, indicating enhanced angiogenesis in mtH–treated wounds. n = 6 per group. (F) Quantitative PCR analysis of angiogenesis-related gene expression in peri-wound tissues on PWD8, indicating transcriptional activation of pro-angiogenic programs following mtH treatment. n = 3 ∼ 5 per group. (G-H) Representative immunohistochemical staining and quantification of Col1a1 in wound tissues on PWD8, showing increased collagen synthesis and matrix remodeling in mtH–treated wounds. n = 6 per group. Scale bar = 100 μm. (I-J) Representative immunofluorescence staining and quantification of Vimentin and TUNEL in wound tissues on PWD8, indicating reduced fibroblast apoptosis following mtH treatment. n = 6 per group. Scale bar = 20 μm. (K-L) Representative immunofluorescence staining and quantification of Vimentin and 8-hydroxyguanosine (8-OHG) in wound tissues on PWD8, indicating attenuated oxidative DNA damage in fibroblasts following mtH treatment. n = 6 per group. Scale bar = 20 μm. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant.

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against CD31 (Servicebio, Cat# GB120005 , 1:200), Vimentin (CST, Cat# 5741, 1:200), and 8-hydroxyguanosine (8-OHG, Rockland, Cat# 200-301-A99, 1:200).

Techniques: In Vivo Imaging, Labeling, Activity Assay, Imaging, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Activation Assay, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: The 84-1 anti-vimentin antibody identifies a distinct form of vimentin in the human brain. a Representative 3D rendering of distinct intracellular vimentin deposits situated within a cell ( a’ ) in 84-1 vimentin-stained brain sections. b and c Vimentin aggregates accumulate within cells (stars) and in S100β-positive astrocytes (box) in the striatum of PD patients ( b ) and the hippocampus of AD patients ( c ). d , Cortical organoids exposed to Aβ 42 soluble aggregates show similar intracellular vimentin deposits that are detected by 84-1vimentin (Magenta), but not with GFAP (green) or Ref. vimentin antibodies (red). Scale bars: 10 μm in ( a ), ( b’ ), ( c’ ), and ( d’ ), 40 μm in ( b ) and ( c ), and 200 μm in ( d )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: 84-1 exhibits a broader labeling pattern and more structural coverage than conventional astrocytic markers. Analysis of human brain Hippocampus (HPC), Caudate Nucleus (CN) and Putamen (PUT), demonstrating the percentage of the non-overlapping structures and the labeling coverage ratio between each co-labeled marker pair: 84-1 (Pink) - GFAP (green), 84-1 (Pink) - S100β (Blue) and 84-1 (Pink) - Ref. vimentin (Violet). n = 5 individuals, Venn diagrams represent the ratio of the mean total area stained and the mean percentage overlap. The full list of analysis results can be found in Supplementary Table S3-5

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Labeling, Marker, Staining

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: The 84-1 antibody labels distinct forms of vimentin in astrocytes and neurons. a The 84-1 antibody identifies astrocytic vimentin, but shows a distinct pattern compared to GFAP and S100β ( a’ ). In addition, it identifies a form of vimentin in neurons ( a’’ ) in the caudate nucleus. b Characteristic pyramidal neurons of the hippocampus stained with the 84-1 antibody display vacuole-like structures within the cytoplasm ( b’ ). Multichannel source images of the rendering are shown in Supplementary Fig. S3. c Brightfield imaging of two examples from human Hippocampus (HPC) and Caudate Nucleus (CN), showing 84-1 vimentin DAB-positive signal. DG: dentate gyrus. d Control staining, following absorption of the 84-1 antibody with recombinant vimentin. Scale bars: 10 μm in ( a ) and ( b ), 20 μm in ( c ) and ( d )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Staining, Imaging, Control, Recombinant

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: Immunoprecipitation analyses of brain homogenates demonstrate that the 84-1 antibody detects cleaved and specifically phosphorylated vimentin forms. a Schematic outline of 84-1 vimentin immunoprecipitation (IP) workflow prior to characterization with Western blot and LC-MS/MS analysis. b Immunoblots of 84-1 IP human brain homogenates (Lysis soluble fraction (S), Lysis insoluble fraction (Ins)) and human CSF. The input blots represent the expression levels of vimentin in the fractions used for IP. IP: Vim 84-1 blots show the experiment output probed with the same IP antibody (IB:84-1) and a Ref. vimentin antibody (IB: Ref. Vim), highlighting a smaller 46 kDa form of vimentin (Box) and clone-specific vimentin bands (Arrow), The full membranes and total protein loading controls are presented in supplementary Fig. S6. c List of the five top proteins detected in the proteomic analysis of the soluble and the insoluble IP isolated fraction, confirming isolation of vimentin. d Detection frequency (y-axis) of semi-tryptic peptide fragments plotted against their position in the vimentin sequence (x-axis). Each bin represents pooled counts from a 20 AA segment of vimentin. Only hits with ≥ 2 counts were included. e Detected Serine (S) and Threonine (T) vimentin phosphorylation in the soluble (S) and insoluble (Ins) fractions, box indicates the spectrum corresponding to the identified peptide with the modified residues highlighted

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Lysis, Expressing, Isolation, Sequencing, Phospho-proteomics, Modification

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: 84-1-vimentin is associated with pathological protein aggregates in the AD and PD brain. a IHC of human PD striatum, using the 84-1 vimentin antibody in combination with the MJF-R13 anti p-αsyn S129 antibody. Arrows indicate characteristic Lewy bodies, and the box highlights 84-1 vimentin overlap. b Co-labelling of 84-1 and the EP1536Y anti p-αsyn S129 antibody. Arrows indicate Lewy bodies/neurites, and boxes represent double-positive aggregates. c Co-labelling of 84-1 and the 81A anti p-αsyn S129 antibody. Arrows indicate characteristic Lewy bodies, and the box represents double-positive aggregates. d IHC of human AD hippocampus displays Aβ 42 deposits around 84-1-vimentin. e - f 84-1-positive staining around dense ( e ) and diffuse Aβ 42 plaques ( f ). Box shows spherical 84-1-vimentin structures in the center of an Aβ 42 plaque, co-localizing with the astrocytic marker GFAP. g - h diffuse 84-1 vimentin expression in neurons carrying AT8 + tangles ( g’ ) and strong astrocytic expression of 84-1-vimentin in regions of high p-Tau231 ( h’ ). Scale bars, 40 μm in ( a - h ), 10 μm in ( a’ - h’ )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Staining, Marker, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: Released 84-1-vimentin represents a potential biomarker for AD and PD pathology. a PLA with the 84-1 vimentin antibody and the EP1536Y p-αsyn S129 antibody revealed positive intracellular and scattered puncta, confirming direct interactions between vimentin and p-αsyn S129 . b Representative ICC images of vimentin 84-1 positive astrocytes ( b’ ) and zombosomes ( b’’ ) in culture, co-stained with the Ref. vimentin antibody. c Immunoblots showing the expression levels of 84-1 vimentin in the soluble and insoluble fraction of cell lysates, as well as in conditioned media from Aβ/αsyn-exposed astrocyte cultures and control cultures. d Western blot quantification of vimentin expression levels, normalized to total proteins for each loaded sample ( n = 5, one-way ANOVA followed by protected Fisher’s LSD post-hoc test. Data shown as mean ± SD. ns: non-significant, * p < 0.05, ** p < 0.01). e and f ELISA analysis of vimentin levels in human CSF samples from PD and age-matched controls ( e ), as well as AD and age-matched controls ( f ), ( n = 7, Two-tailed unpaired t-test. Data shown as mean ± SD. * p < 0.05). The full membranes and total protein loading controls are shown in Supplementary Fig. S7. Scale bars: 20 μm in ( a ), ( b ), 10 μm in ( b’ ), ( b’’ )

Article Snippet: CSF samples were first boiled in 1mM DTT at 95 °C for 10 min. Recombinant human vimentin (FisherScientific, 17810393) was used as a standard.

Techniques: Biomarker Discovery, Staining, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: The 84-1 anti-vimentin antibody identifies a distinct form of vimentin in the human brain. a Representative 3D rendering of distinct intracellular vimentin deposits situated within a cell ( a’ ) in 84-1 vimentin-stained brain sections. b and c Vimentin aggregates accumulate within cells (stars) and in S100β-positive astrocytes (box) in the striatum of PD patients ( b ) and the hippocampus of AD patients ( c ). d , Cortical organoids exposed to Aβ 42 soluble aggregates show similar intracellular vimentin deposits that are detected by 84-1vimentin (Magenta), but not with GFAP (green) or Ref. vimentin antibodies (red). Scale bars: 10 μm in ( a ), ( b’ ), ( c’ ), and ( d’ ), 40 μm in ( b ) and ( c ), and 200 μm in ( d )

Article Snippet: To confirm signal specificity, an absorption control was performed by pre-incubating the 84-1 antibody with a ten-fold molar excess of recombinant vimentin (Fisher Scientific, 17810393) at 4o C ON prior to staining.

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: 84-1 exhibits a broader labeling pattern and more structural coverage than conventional astrocytic markers. Analysis of human brain Hippocampus (HPC), Caudate Nucleus (CN) and Putamen (PUT), demonstrating the percentage of the non-overlapping structures and the labeling coverage ratio between each co-labeled marker pair: 84-1 (Pink) - GFAP (green), 84-1 (Pink) - S100β (Blue) and 84-1 (Pink) - Ref. vimentin (Violet). n = 5 individuals, Venn diagrams represent the ratio of the mean total area stained and the mean percentage overlap. The full list of analysis results can be found in Supplementary Table S3-5

Article Snippet: To confirm signal specificity, an absorption control was performed by pre-incubating the 84-1 antibody with a ten-fold molar excess of recombinant vimentin (Fisher Scientific, 17810393) at 4o C ON prior to staining.

Techniques: Labeling, Marker, Staining

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: The 84-1 antibody labels distinct forms of vimentin in astrocytes and neurons. a The 84-1 antibody identifies astrocytic vimentin, but shows a distinct pattern compared to GFAP and S100β ( a’ ). In addition, it identifies a form of vimentin in neurons ( a’’ ) in the caudate nucleus. b Characteristic pyramidal neurons of the hippocampus stained with the 84-1 antibody display vacuole-like structures within the cytoplasm ( b’ ). Multichannel source images of the rendering are shown in Supplementary Fig. S3. c Brightfield imaging of two examples from human Hippocampus (HPC) and Caudate Nucleus (CN), showing 84-1 vimentin DAB-positive signal. DG: dentate gyrus. d Control staining, following absorption of the 84-1 antibody with recombinant vimentin. Scale bars: 10 μm in ( a ) and ( b ), 20 μm in ( c ) and ( d )

Article Snippet: To confirm signal specificity, an absorption control was performed by pre-incubating the 84-1 antibody with a ten-fold molar excess of recombinant vimentin (Fisher Scientific, 17810393) at 4o C ON prior to staining.

Techniques: Staining, Imaging, Control, Recombinant

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: Immunoprecipitation analyses of brain homogenates demonstrate that the 84-1 antibody detects cleaved and specifically phosphorylated vimentin forms. a Schematic outline of 84-1 vimentin immunoprecipitation (IP) workflow prior to characterization with Western blot and LC-MS/MS analysis. b Immunoblots of 84-1 IP human brain homogenates (Lysis soluble fraction (S), Lysis insoluble fraction (Ins)) and human CSF. The input blots represent the expression levels of vimentin in the fractions used for IP. IP: Vim 84-1 blots show the experiment output probed with the same IP antibody (IB:84-1) and a Ref. vimentin antibody (IB: Ref. Vim), highlighting a smaller 46 kDa form of vimentin (Box) and clone-specific vimentin bands (Arrow), The full membranes and total protein loading controls are presented in supplementary Fig. S6. c List of the five top proteins detected in the proteomic analysis of the soluble and the insoluble IP isolated fraction, confirming isolation of vimentin. d Detection frequency (y-axis) of semi-tryptic peptide fragments plotted against their position in the vimentin sequence (x-axis). Each bin represents pooled counts from a 20 AA segment of vimentin. Only hits with ≥ 2 counts were included. e Detected Serine (S) and Threonine (T) vimentin phosphorylation in the soluble (S) and insoluble (Ins) fractions, box indicates the spectrum corresponding to the identified peptide with the modified residues highlighted

Article Snippet: To confirm signal specificity, an absorption control was performed by pre-incubating the 84-1 antibody with a ten-fold molar excess of recombinant vimentin (Fisher Scientific, 17810393) at 4o C ON prior to staining.

Techniques: Immunoprecipitation, Western Blot, Liquid Chromatography with Mass Spectroscopy, Lysis, Expressing, Isolation, Sequencing, Phospho-proteomics, Modification

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: 84-1-vimentin is associated with pathological protein aggregates in the AD and PD brain. a IHC of human PD striatum, using the 84-1 vimentin antibody in combination with the MJF-R13 anti p-αsyn S129 antibody. Arrows indicate characteristic Lewy bodies, and the box highlights 84-1 vimentin overlap. b Co-labelling of 84-1 and the EP1536Y anti p-αsyn S129 antibody. Arrows indicate Lewy bodies/neurites, and boxes represent double-positive aggregates. c Co-labelling of 84-1 and the 81A anti p-αsyn S129 antibody. Arrows indicate characteristic Lewy bodies, and the box represents double-positive aggregates. d IHC of human AD hippocampus displays Aβ 42 deposits around 84-1-vimentin. e - f 84-1-positive staining around dense ( e ) and diffuse Aβ 42 plaques ( f ). Box shows spherical 84-1-vimentin structures in the center of an Aβ 42 plaque, co-localizing with the astrocytic marker GFAP. g - h diffuse 84-1 vimentin expression in neurons carrying AT8 + tangles ( g’ ) and strong astrocytic expression of 84-1-vimentin in regions of high p-Tau231 ( h’ ). Scale bars, 40 μm in ( a - h ), 10 μm in ( a’ - h’ )

Article Snippet: To confirm signal specificity, an absorption control was performed by pre-incubating the 84-1 antibody with a ten-fold molar excess of recombinant vimentin (Fisher Scientific, 17810393) at 4o C ON prior to staining.

Techniques: Staining, Marker, Expressing

Journal: Acta Neuropathologica Communications

Article Title: Characterization of a distinct form of vimentin in the neurodegenerative brain

doi: 10.1186/s40478-026-02324-9

Figure Lengend Snippet: Released 84-1-vimentin represents a potential biomarker for AD and PD pathology. a PLA with the 84-1 vimentin antibody and the EP1536Y p-αsyn S129 antibody revealed positive intracellular and scattered puncta, confirming direct interactions between vimentin and p-αsyn S129 . b Representative ICC images of vimentin 84-1 positive astrocytes ( b’ ) and zombosomes ( b’’ ) in culture, co-stained with the Ref. vimentin antibody. c Immunoblots showing the expression levels of 84-1 vimentin in the soluble and insoluble fraction of cell lysates, as well as in conditioned media from Aβ/αsyn-exposed astrocyte cultures and control cultures. d Western blot quantification of vimentin expression levels, normalized to total proteins for each loaded sample ( n = 5, one-way ANOVA followed by protected Fisher’s LSD post-hoc test. Data shown as mean ± SD. ns: non-significant, * p < 0.05, ** p < 0.01). e and f ELISA analysis of vimentin levels in human CSF samples from PD and age-matched controls ( e ), as well as AD and age-matched controls ( f ), ( n = 7, Two-tailed unpaired t-test. Data shown as mean ± SD. * p < 0.05). The full membranes and total protein loading controls are shown in Supplementary Fig. S7. Scale bars: 20 μm in ( a ), ( b ), 10 μm in ( b’ ), ( b’’ )

Article Snippet: To confirm signal specificity, an absorption control was performed by pre-incubating the 84-1 antibody with a ten-fold molar excess of recombinant vimentin (Fisher Scientific, 17810393) at 4o C ON prior to staining.

Techniques: Biomarker Discovery, Staining, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test