recombinant human vimentin  (Fisher Scientific)

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

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner ( n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [ n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676 .]

Article Snippet: For experiments involving binding of ROME-FL to vimentin (Novus Biologicals, #NBP2-35139-100 μg) and β-catenin (Novus Biologicals, #NBP3-18198-100 μg), vimentin and β-catenin were immobilized as ligands onto CM5 chip surfaces.

Techniques: Binding Assay, Recombinant, Injection, Expressing, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control

Journal: The Journal of Biological Chemistry

Article Title: HSP-1-specific nanobodies alter chaperone function in vitro and in vivo

doi: 10.1016/j.jbc.2026.111238

Figure Lengend Snippet: B12 and H5 detect HSP-1 from C. elegans’ lysate. A and B , western blots of day 2 C. elegans that were grown on control ( pos-1 RNAi) plates and then transferred to either pos-1 or hsp-1 RNAi plates for 24 h as day 1 adults. pos-1 encodes a zinc-finger transcription factor required for embryonic development and sterilizes worms without affecting hatched animals ( , , , , , ). Membranes were probed with either biotinylated ( A ) B12 or ( B ) H5 and α-tubulin (DSHB, Cat #12G10). C , western blots from immunoprecipitation assay using recombinant H5 or B12 as the primary antigen-binding agent and probed with an anti-HSC70 antibody (Proteintech, Cat #10654-1-AP) and α-tubulin (DSHB, Cat #12G10). D and E , example ( D ) western blot and ( E ) quantification of nanobody B12 expression (MTX298 ( hsp-16.48p::B12::HA )) induced by a 30-min heat shock at 37 °C (HA, Cell Signaling (C29F4) and α-tubulin (DSHB, Cat #12G10)). F and G , example ( F ) western blot and ( G ) quantification of nanobody B12 expression (MTX298 ( hsp-16.48p::B12::HA )) placed at 25 °C for indicated amount of time or induced by 30-min heat shock at 37 °C (HA, Cell Signaling (C29F4) and α-tubulin (DSHB, Cat #12G10). Induction was started in day 1 adults. H , co-immunoprecipitation assay of WT and MTX298 (“B12”) grown on indicated siRNA and collected 5 h post 30-min heat shock at 37 °C using magnetic anti-HA beads. Ordinary one-way ANOVA. p < 0.05 is considered statistically significant. HSP, heat shock protein; HA, hemagglutinin; HSC70, heat shock cognate 70.

Article Snippet: C , western blots from immunoprecipitation assay using recombinant H5 or B12 as the primary antigen-binding agent and probed with an anti-HSC70 antibody (Proteintech, Cat #10654-1-AP) and α-tubulin (DSHB, Cat #12G10).

Techniques: Western Blot, Control, Immunoprecipitation, Recombinant, Binding Assay, Expressing, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: TRIM44 was down-regulated in clear cell renal cell carcinoma (ccRCC) and associated with disease progression. A , Heatmap representation of comparative proteomic analysis between primary (786-O, 769-P, OSRC-2) and metastatic (ACHN, Caki-1, SN12-PM6) ccRCC cell lines. Fold change> 1.2 or <0.83, p < 0.05 was regarded as significant. B , Volcano plot demonstrating significant downregulation of TRIM44 in metastatic versus primary ccRCC cell lines. C , Expression intensity of endogenous TRIM44 in six ccRCC cell lines and statistical data graph ( ∗ compared with 786-O, # compared with 769-P). D , Representative IHC staining of TRIM44, vimentin, E-Cadherin and Ki-67 in ccRCC tissues. Scale bar: 50 μm. E , statistical data graph of TRIM44 expression in peritumour and different tumor stages ( ∗ compared with Paritumour, # compared with StageⅠ-Ⅱ). F , expression of TRIM44 and vimentin in 12 pairs of ccRCC samples. G , Kaplan–Meier survival analysis showing poorer overall survival in TRIM44-low versus TRIM44-high ccRCC patients (HMU-RCC cohort). H , combined survival analysis of TRIM44 and vimentin expression levels. ∗ p < 0.05, ∗∗ p < 0.01 and ## p < 0.01.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: Biomarker Discovery, Expressing, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: TRIM44 restrained ccRCC progression in vitro . A – D , knockdown of TRIM44 enhanced cell migration, invasion, proliferation, and colony formation ability in 769-P cell lines; overexpression of TRIM44 reduced cell migration, invasion, proliferation, and colony formation ability in Caki-1 cell lines. A , cell wound scratch assay. Scale bar: 500 μm. B , Transwell migration and invasion assay. Scale bar: 100 μm. C , EdU proliferation staining, Scale bar: 25 μm. D , Colony formation assay. E and F , the expression of E-Cadherin, vimentin, snai1, and TRIM44 was detected by Western blot assay. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: In Vitro, Knockdown, Migration, Over Expression, Wound Healing Assay, Invasion Assay, Staining, Colony Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: TRIM44 is bound to vimentin in the cytoplasm in ccRCC cells. A and B , identification of the interacting protein of TRIM44 by a combination of Co-IP and MS in 769-P and Caki-1 cell lines. C and D , Validation of TRIM44-vimentin interaction by Co-IP assays in 769-P and Caki-1 cell lines. E , IF analysis demonstrating co-localization of TRIM44 and vimentin in 769-P and Caki-1 cell lines. Scale bar: 25 μm. F and G , the interaction of TRIM44 and vimentin was tested by Co-IP assay in the cytoplasm and nucleus, respectively. H , PLA assay was performed to analyze the association of TRIM44/vimentin ( red ) in 769-P and Caki-1 cell lines; the scatter plot showed the average PLA dots per cell. Scale bar: 5 μm. ∗∗ p < 0.01.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: Co-Immunoprecipitation Assay, Biomarker Discovery

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: TRIM44 negatively regulated vimentin by promoting its degradation via ubiquitination. A and B , the mRNA level of vimentin was detected by RT-PCR after TRIM44 interference in 769-P and Caki-1 cells. C–E , the expression of vimentin protein was examined by Western blot. C and D , 769-P cells were transfected with shNC or shTRIM44 plasmid, and 10 μM MG132 for 8 h. E , Caki-1 cells were transfected with Vector or TRIM44 plasmid and added 10 μM MG132 for 8 h. F–H , the expression level of vimentin was detected by Western blot assay. F and G , 769-P cells were transfected with shNC or shTRIM44 plasmid and treated with 100 μg/ml CHX. H, Caki-1 cells were transfected with the Vector or TRIM44 plasmid and treated with 100 μg/ml CHX. And line charts of the degradation rate of vimentin in 769-P and Caki-1 cell lines. ∗ p < 0.05; ∗∗ p < 0.01 and NS, no significance.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: TRIM44 targeted vimentin for K48-linked polyubiquitination. A and B , transfecting shNC or shTRIM44 plasmid to 769-P cells, vector or TRIM44 plasmid to Caki-1 cells, and Ub plasmid and MG132 (10 μM for 8 h) to all above cells. The lysates of cells were immunoprecipitated with vimentin antibody and performing Western blot assay with Ub antibody. C , HEK-293T cells were co-transfected with vector/TRIM44, vimentin and Ub plasmids, and MG132 (10 μM for 8 h). The lysates of cells were immunoprecipitated with vimentin antibody and performing Western blot assay with Ub antibody. D , the schematic diagram of TRIM44 mutant protein. Full-length (FL) TRIM44 (amino acids 1–344); ZF domain, a construct amino acids 13 to 48; BB domain, a construct amino acids 174 to 215; CC domain, a construct amino acids 290 to 325; TRIM44 with a deleted BB domain (△B), a construct without the BB domain (del 174–215). E , HEK-293T cells were co-transfected with HA-vimentin and MYC-TRIM44 or TRIM44 deletion mutants. The lysates of cells were immunoprecipitated with MYC-tag antibody and performing Western blot assay with HA-tag antibody. F , molecular docking ( http://hdock.phys.hust.edu.cn ) to predict the binding site between TRIM44 (in rainbow color) and vimentin (in yellow color). G , the in vitro ubiquitylation assay was performed by using purified TRIM44-BB, TRIM44-ΔB and vimentin protein in the presence of E1, E2, ubiquitin and ATP. Western blotting was applied to examine the levels of ubiquitin. H and I , HEK-293T cells were co-transfected with MYC-TRIM44-BB, HA-vimentin, Flag-Ub-K48/K63 and MG132 (10 μM for 8 h). H , the lysates of cells were immunoprecipitated with HA-tag antibody and performing Western blot assay with Ub antibody. I , the lysates of cells were immunoprecipitated with Flag-tag antibody and performing Western blot assay with HA antibody. J and K , HEK-293Tcells were transfected with Vector or mutated plasmid with MYC-TRIM44-BB, HA-vimentin and Ub-K48 plasmid ( J ) or Ub-K63 plasmid ( K ), and treated with 100 μg/ml CHX.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Construct, Binding Assay, In Vitro, Ubiquitin Assay, Purification, Ubiquitin Proteomics, FLAG-tag

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: TRIM44 correlated with vimentin and affected the progression of ccRCC in vivo . A , lung images of nude mice through tail vein injection of shNC or shTRIM44 derived 769-P cells, n = 5 of each group. And lung images were performed to detect metastatic foci. B , statistical data graph of lung metastasis quantification. C , representative H&E staining of lung sections. Scale bar: 100 μm. D , The expression of vimentin, E-Cadherin, and Ki-67 was edtected by IHC in ccRCC mouse model. Scale bar: 100 μm. ∗∗ p < 0.01.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: In Vivo, Injection, Derivative Assay, Staining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Loss of TRIM44 promotes renal cell carcinoma progression by regulating K48-linked ubiquitination of vimentin

doi: 10.1016/j.jbc.2025.110734

Figure Lengend Snippet: Schematic model illustrating the mechanisms of TRIM44-mediated vimentin ubiquitination regulated EMT and tumor metastasis in ccRCC.

Article Snippet: Recombinant TRIM44-BB protein (GENEWIZ) and TRIM44-ΔB protein (GENEWIZ) used in this study were commercially synthesized and produced in a mammalian expression system (HEK293T cells), while recombinant vimentin protein (Sino Biological, 10,028-H08 B) was produced in the baculovirus system (Sf9 insect cells).

Techniques: Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Vaccination Against Extracellular Vimentin Plus Doxorubicin for Canine Hemangiosarcoma

doi: 10.3390/ijms26189096

Figure Lengend Snippet: Immunohistochemistry, ultrasound and CT of canine HSA. ( A ) Representative images of HSA of two different dogs of Hematoxylin/eosin (HE) staining and staining for ETS-related Gene (ERG), CD45 and vimentin are shown. Scale bar in lower right panel represents 100 μm. ( B ) Abdominal ultrasound image of a dog with a primary splenic HSA. Yellow cross indicates the tumor dimensions. ( C ) Ultrasound image of the same dog showing liver metastases developed on day 276 post-surgery, indicated by yellow arrowheads. ( D ) Thoracic radiograph of a dog with splenic HSA, showing abundant lung metastases.

Article Snippet: Antibody titers were measured in duplicate in 96-well ELISA plates (F96 Maxisorp, Nunc A/S, Roskilde, Denmark) coated with 4 μg/mL recombinant dog vimentin protein and subsequently blocked with 4% milk/PBS (100 μL/well) (sc-2325, Santa Cruz Biotechnology, Dallas, TX, USA), both for 1 h at 37 °C.

Techniques: Immunohistochemistry, Staining