Journal: Clinical and Experimental Immunology

Article Title: Anti‐vimentin/cardiolipin IgA in the anti‐phospholipid syndrome: A new tool for ‘seronegative’ diagnosis

doi: 10.1111/cei.13633

Figure Lengend Snippet: Prevalence of antibodies specific for vimentin/cardiolipin complex.

Article Snippet: Briefly, a 96‐well polystyrene plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was coated with 100 μl/well of cardiolipin (50 μg/ml in methanol) (from bovine heart; Sigma‐Aldrich, St Louis, Missouri, USA) and human recombinant vimentin (5 μg/ml in 0.05 mM NaHCO 3 buffer, pH 9.5) (R&D Systems, Minneapolis, Minnesota, USA).

Techniques:

Journal: Clinical and Experimental Immunology

Article Title: Anti‐vimentin/cardiolipin IgA in the anti‐phospholipid syndrome: A new tool for ‘seronegative’ diagnosis

doi: 10.1111/cei.13633

Figure Lengend Snippet: Levels of anti‐vimentin/cardiolipin (aVim/CL) immunoglobulin (Ig)A in patients [anti‐phospholipid syndrome (APS), seronegative (SN)‐APS] and in healthy controls (HC). For detection of aVim/CL IgA all the sera were analyzed by enzyme‐linked immunosorbent assay (ELISA). The cut‐off level has been calculated as the 99th percentile of 40 HC sera

Article Snippet: Briefly, a 96‐well polystyrene plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was coated with 100 μl/well of cardiolipin (50 μg/ml in methanol) (from bovine heart; Sigma‐Aldrich, St Louis, Missouri, USA) and human recombinant vimentin (5 μg/ml in 0.05 mM NaHCO 3 buffer, pH 9.5) (R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Anti‐vimentin/cardiolipin IgA in the anti‐phospholipid syndrome: A new tool for ‘seronegative’ diagnosis

doi: 10.1111/cei.13633

Figure Lengend Snippet: Distribution of positive seronegative‐anti‐phospholipid syndrome (SN‐APS) patients among those tested positive for at least one immunoglobulin (Ig)A assay: anti‐cardiolipin (aCL), anti‐β2‐glycoprotein I (aβ2‐GPI); anti‐vimentin/cardiolipin (aVim/CL)

Article Snippet: Briefly, a 96‐well polystyrene plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was coated with 100 μl/well of cardiolipin (50 μg/ml in methanol) (from bovine heart; Sigma‐Aldrich, St Louis, Missouri, USA) and human recombinant vimentin (5 μg/ml in 0.05 mM NaHCO 3 buffer, pH 9.5) (R&D Systems, Minneapolis, Minnesota, USA).

Techniques:

Journal: Clinical and Experimental Immunology

Article Title: Anti‐vimentin/cardiolipin IgA in the anti‐phospholipid syndrome: A new tool for ‘seronegative’ diagnosis

doi: 10.1111/cei.13633

Figure Lengend Snippet: Receiver operating characteristic (ROC) analysis of seronegative‐anti‐phospholipid syndrome (SN‐APS). A Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI‐2K) variation presented an area under the curve (AUC) of 0.697 [95% confidence interval (CI) = 0.628–0.766, p < 0.0005]. The variation in anti‐vimentin/cardiolipin (aVim/CL) immunoglobulin (Ig)A levels presented an area under the curve (AUC) of 0.75 (95% CI = 0.645–0.856, p < 0.0001, Youden’s J = 0.213) for the development of thrombotic or pregnancy events, as indicated in the APS classification criteria

Article Snippet: Briefly, a 96‐well polystyrene plate (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was coated with 100 μl/well of cardiolipin (50 μg/ml in methanol) (from bovine heart; Sigma‐Aldrich, St Louis, Missouri, USA) and human recombinant vimentin (5 μg/ml in 0.05 mM NaHCO 3 buffer, pH 9.5) (R&D Systems, Minneapolis, Minnesota, USA).

Techniques: Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: The SMRwt peptide specifically interacts with host cell proteins, including Mortalin and Vimentin. ( A ) Proteins from MDA-MB-231, MCF-7, and BT474 BC cells were co-immunoprecipitated with either SMRwt or SMRmut peptides using an anti-FLAG M2 antibody-coupled affinity resin. The procedure was also performed without the peptides as a control for nonspecific interactions. ( B ) Mortalin and ( C ) Vimentin. Densitometric analysis of Western blot data using the NIH ImageJ software 1.52 (NIH, Bethesda, MD, USA) (due to its sensitivity to pixel density and background correction) revealed significant differences: SMRmut vs. SMRwt **** p < 0.00005 in MDA-MB-231, **** p < 0.00005 in MCF-7, and **** p < 0.00005 in BT474 cells when probed with a Mortalin (Grp-75) antibody; **** p < 0.00005 in MDA-MB-231, **** p < 0.00005 in MCF-7, and **** p < 0.00005 in BT474 cells when probed with a Vimentin antibody.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Immunoprecipitation, Control, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Differences between WT (wild-type) and MUT (mutant) peptides. (A) SMR peptides (HIV-1 Nef protein sequence has 206 AA): Schematic representation of the wild-type (SMRwt) and negative-control (SMRmut) synthetic peptides derived from HIV-1 Nef (206 AA) . The difference: The first amino acid in the sequence changes from valine (V) in the WT peptide to alanine (A) in the MUT peptide. (B) SMR-FLAGtag peptides (HIV-1 Nef protein sequence has 206 AA): Schematic representation of the wild-type (SMRwt) and negative-control (SMRmut) synthetic peptides derived from HIV-1 Nef (206 AA) , showing sites of amino acid changes in SMRmut. Each peptide contains a FLAG tag at the C-terminus. The difference between SMRwt-FLAGtag and SMRmut-FLAGtag is that SMRwt-FLAGtag contains a valine (V) at the first position, while SMRmut-FLAGtag contains an alanine (A) at the first position. (C) Mortalin peptides: The Mortalin protein sequence has 679 AA. The sequences of Mortalin #61 and #62 show regions implicated in binding the HIV-1 Nef and SMRwt peptide. Mortalin #56 did not bind to Nef or the SMRwt peptide. The difference: These peptides are entirely different sequences, each representing different segments of the Mortalin protein. (D) Vimentin peptides: The Vimentin sequence has 466 AA. The difference between the Vimentin-wt peptide (LYEEEMRE) and the Vimentin-mut peptide (LYEEEMRE, red) lies in the substitution of the sequence YEEEM with AAAAA in the mutant peptide. This change significantly alters the peptide’s structure and function.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Mutagenesis, Sequencing, Negative Control, Derivative Assay, FLAG-tag, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Effects of SMR, Mortalin, and Vimentin peptides on tumor cell exosome secretion in MDA-MB-231 and MCF-7 breast cancer cells. MDA-MB-231 and MCF-7 BC cells (200 µL, 1 × 10 4 cells/mL) were seeded into a 96-well plate and treated with 100 ng/mL of either wild-type (wt) or mutant (mut) peptides of SMR, Mortalin, or Vimentin. Treatments were performed in RPMI-1640 medium without transfection, and cells were incubated at 37 °C for 24 h. The cells were then treated with 0.5 µM/mL of N-Rh-PE, incubated at 4 °C for 1 h, and washed with PBS. Fresh medium was also added. The cultures were incubated at 37 °C for an additional 24 h. N-Rh-PE refers to N-(Lissamine Rhodamine B sulfonyl) phosphatidyl-ethanolamine, a fluorescent lipid dye commonly used to label membranes and track vesicle trafficking. ( A ) MDA-MB-231 Cells: Data analysis of exosome release revealed significant differences: UT vs. SMRwt, Mort #61/62(B)-wt, VIM-wt **** p < 0.00005 in MDA-MB-231. ( B ) MCF-7 Cells: Data analysis of exosome release revealed significant differences: UT vs. SMRwt, Mort #61/62(B)-wt, VIM-wt **** p < 0.00005 in MCF-7. Exosome secretion was assessed by measuring the amount of N-Rh-PE, which corresponds to the number of exosomes released into the extracellular medium. Bar graphs show the relative exosome release level. Data represent the mean ± SD of three independent experiments.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Mutagenesis, Transfection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: SPR analysis of SMRwt, SMRmut, and Nef binding to Vimentin and Mortalin. ( A ) Binding of SMRwt ( left ) and SMRmut ( right ) to immobilized Vimentin. ( B ) Binding of SMRwt ( left ) and SMRmut ( right ) to immobilized Mortalin. ( C ) Binding of Nef protein to immobilized Vimentin ( left ) and Mortalin ( right ). Colored lines represent experimental data, while black lines indicate fits to a 1:1 kinetics binding model. The derived kinetic parameters are displayed. Three independent experiments were performed, with one representative run shown here.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Binding Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Competitive binding of SMR and Mortalin peptides to Vimentin and Mortalin proteins. Binding of SMRwt to immobilized Vimentin in the presence and absence of ( A ) Mortalin peptide #56, ( B ) Mortalin peptide #61, and ( C ) Mortalin peptide #62. Binding of SMRwt to immobilized Mortalin in the presence and absence of ( D ) Mortalin peptide #56, ( E ) Mortalin peptide #61, and ( F ) Mortalin peptide #62. SMRwt alone, as well as a mixture of His-tagged Mortalin peptides #56, #61, and #62 were injected as analytes to flow over the immobilized ligand surfaces. ( B , E ) show that Mortalin peptide #61 completely blocks the 20 µM SMRwt from binding to both Vimentin and Mortalin on the surface. By contrast, Mortalin peptide #56 ( A , D ) provides only partial blockage at 20 µM. In contrast to ( F ) for SMRwt binding to Mortalin, ( C ) shows no blockage of SMRwt binding to Vimentin. Vimentin and Mortalin proteins were immobilized as ligands on a CM5 sensor chip using standard amine coupling chemistry.

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Binding Assay, Injection

Journal: International Journal of Molecular Sciences

Article Title: Investigating SMR Peptide Interactions with Breast Cancer-Associated Proteins

doi: 10.3390/ijms26188848

Figure Lengend Snippet: Binding scenarios involving Mortalin peptides competing with the SMR peptide, and the resulting effects on interactions between SMRwt and either Vimentin or Mortalin. ( A ) Full Nef protein or SMR peptide interactions with target proteins. ( B ) Full Nef protein + SMR peptide in competition for binding to target protein. ( C ) Full Nef protein or SMRwt peptide in competition with the Mortalin binding site for binding to target protein. Created with BioRender Brena, D (2025). https://BioRender.com/042t0rb (accessed on 5 September 2025).

Article Snippet: Recombinant Human Vimentin Protein, CF (catalog # 2105-VI-100), Recombinant Human GRP75/HSPA9B/Mortalin His Protein (catalog # NBC1-18380), and Recombinant Human HSPA8/HSC71/Hsc70 His Protein (Catalog # NBPI-30278) were purchased from Novus Biologicals, LLC/R&D Systems, Centennial, CO, USA.

Techniques: Binding Assay

Journal: Antioxidants

Article Title: Molecular Insight into the Regulation of Vimentin by Cysteine Modifications and Zinc Binding

doi: 10.3390/antiox10071039

Figure Lengend Snippet: Vimentin dimer and its interaction with zinc. ( A ) Zinc binding at vimentin B dimer. Filled and ribbon views of dimer B are shown. After 100 ns MD simulation, zinc atoms, shown as orange spheres, were placed in electronegative regions. Below, the region near Cys328 is displayed in detail, illustrating the interaction of zinc atoms with Glu329 and Asp331. After 30 ns of MD simulation, two zinc atoms remained close to Cys328, interacting with Glu329 and Asp331 during the whole simulation time. ( B ) Zinc binding at vimentin B dimer, with Cys328 in its thiolate form. In this case, the cysteine interacts directly with zinc atoms, as displayed in the detailed view. ( C ) Experimental determination of vimentin Cys328 pK a . Kinetics of MBB modification of vimentin wild type (wt, left panel) and Cys328Ser mutant (right panel) were measured at different pHs in triplicate using a Varioskan plate reader and the k obs calculated for each case. The pK a was obtained from the representation of k obs values against the final pH of the reaction. ( D ) Protection of vimentin modification by zinc. Purified vimentin wt (4.3 μM final concentration) was incubated with ZnCl 2 at the indicated concentrations for 1 h at r.t., before adding vehicle, biotinylated iodoacetamide (10 μM, Iac-B, left panel), or biotinylated 15d-PGJ 2 (1 μM, 15d-PGJ 2 -B, right panel), and incubating an additional hour at 37 °C. Incubation mixtures were run on SDS-PAGE gels, electro-blotted, and incorporation of the biotinylated compounds assessed by biotin detection with HRP-conjugated streptavidin. Vimentin was detected by western blot. Dotted lines indicate where lanes from the same gel have been cropped. The position of molecular weight standards (in kDa) is indicated on the left. The ratio of the biotin and the vimentin signals is displayed in the graph as mean values ± SEM of four independent assays. * p < 0.05 vs. Iac-B or 15d-PGJ 2 -B at 0 μM ZnCl 2 ; *** p < 0.0005 vs. Iac-B or 15d-PGJ 2 -B at 0 μM ZnCl 2 by Student’s t -test.

Article Snippet: Recombinant human vimentin wild type (wt) and Cys328Ser in 8 M urea, 5 mM Tris-HCl pH 7.6, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.4 mM PMSF, and approximately 0.2 M KCl, purified essentially as described [ ], were purchased from Biomedal (Spain).

Techniques: Binding Assay, Modification, Mutagenesis, Purification, Concentration Assay, Incubation, SDS Page, Western Blot, Molecular Weight

Journal: Antioxidants

Article Title: Molecular Insight into the Regulation of Vimentin by Cysteine Modifications and Zinc Binding

doi: 10.3390/antiox10071039

Figure Lengend Snippet: Dimer B of a vimentin Cys328Ser mutant and its interaction with zinc. ( A ) Final snapshot of a 100 ns MD simulation of the Cys328Ser mutant in water. ( B ) Final snapshot of 100 ns MD simulation of this mutant in the presence of zinc. In both cases, lower panels show an amplified view of the region around Ser328. Zinc atoms are represented as orange spheres.

Article Snippet: Recombinant human vimentin wild type (wt) and Cys328Ser in 8 M urea, 5 mM Tris-HCl pH 7.6, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.4 mM PMSF, and approximately 0.2 M KCl, purified essentially as described [ ], were purchased from Biomedal (Spain).

Techniques: Mutagenesis, Amplification

Journal: Antioxidants

Article Title: Molecular Insight into the Regulation of Vimentin by Cysteine Modifications and Zinc Binding

doi: 10.3390/antiox10071039

Figure Lengend Snippet: Cysteine-crosslinking of vimentin and potential conformations of the resulting dimers of dimers obtained by molecular dynamics. ( A ) Cysteine-crosslinking of vimentin (4.7 μM final concentration) upon incubation with vehicle, 1 mM diamide, 24 μM DBB, or 1 mM H 2 O 2 for 1 h at r.t. Incubation mixtures were analyzed by SDS-PAGE under non-reducing conditions and western blot. The position of the oligomer detected under these conditions is marked by an arrow. The oligomer/monomer ratios are displayed below the plot as average values ± SEM of three independent experiments. ( B ) Cys328 is required for DBB crosslinking. Vimentin wt or Cys328Ser (4.5 μM final concentration) were incubated with 50 μM DBB for 1 h at r.t, and subjected to SDS-PAGE, followed by Sypro Ruby staining for detection of vimentin monomeric and oligomeric (marked by a bracket) species under UV light. Cysteine-crosslinked oligomeric species were estimated by image scanning, and values are presented below the plot in arbitrary units. ( C ) Effect of NaCl-induced polymerization on DBB vimentin crosslinking. Vimentin wt (3.8 μM final concentration) was incubated with vehicle or 150 mM NaCl for 10 min at 37 °C (conditions that induce polymerization) and subsequently incubated for 1 h at r.t. with vehicle or 24 μM DBB. Samples were analyzed by SDS-PAGE and western blot, and representative results are presented. The proportion of oligomeric (bracket) versus monomeric species is shown in the graph on the right. Results are average values of three assays ± SEM. * p < 0.05 vs. control (-NaCl, -DBB); # p < 0.05 vs. (-NaCl, +DBB) by paired Student’s t -test. ( D ) Models of vimentin dimers of dimers. Distant-CYS and nearby-CYS tetramers are represented in orange and blue, respectively. For each dimer, 100 ns of MD simulation were performed. The structures of the initial and last frames of the simulation are shown. Cys328 is represented in spheres. ( E ) Front view of the dimerization interface between both vimentin dimers in the nearby-CYS tetramer. ( F , G ) Semi-lateral ( F ) and lateral ( G ) views of both vimentin dimers in the nearby-CYS tetrameric association with details of the interactions that take place at the dimerization interface. Interacting residues are depicted as sticks.

Article Snippet: Recombinant human vimentin wild type (wt) and Cys328Ser in 8 M urea, 5 mM Tris-HCl pH 7.6, 1 mM EDTA, 10 mM 2-mercaptoethanol, 0.4 mM PMSF, and approximately 0.2 M KCl, purified essentially as described [ ], were purchased from Biomedal (Spain).

Techniques: Concentration Assay, Incubation, SDS Page, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Extracellular vimentin as a target against SARS-CoV-2 host cell invasion

doi: 10.1101/2021.01.08.425793

Figure Lengend Snippet: Purified human recombinant vimentin was added to suspensions of SARS-CoV-2 spike protein-containing pseudovirus, and their size was measured by dynamic light scattering (a) and atomic force microscopy (b-e). The size of the pseudovirus increased from 60 nm to 150 nm after addition of 0.07 mg/ml vimentin (panel a). Panel b-d: The pseudoviruses were imaged using atomic force microscopy before and after addition of either DNA (0.08 mg/mL) or vimentin (0.07 mg/ml). The probability distribution functions (e) show that the average size of pseudovirus imaged by AFM confirms the change in size detected by DLS. (f) Schematic representation indicating how binding of vimentin to SARS-CoV-2 spike protein could couple particles together and increase their effective radii.

Article Snippet: Briefly, for AFM evaluation of pseudoviruses in control (non-vimentin addition) and samples preincubated with human recombinant vimentin, a 10 μl drop of pseudoviruses or pseudoviruses/vimentin was applied on cleaved mica (SPI Supplies, West Chester, PA) and analyzed in a dried environment.

Techniques: Purification, Recombinant, Microscopy, Binding Assay

Journal: bioRxiv

Article Title: Extracellular vimentin as a target against SARS-CoV-2 host cell invasion

doi: 10.1101/2021.01.08.425793

Figure Lengend Snippet: ( a ) Extracellular vimentin was detected on A549-hsACE2 as shown by immunofluorescence images, ( b ) The recombinant Anti-Vimentin antibody - rabbit monoclonal IgG, which binds to the vimentin C-terminus blocks uptake in both HEK 293T-hsACE2 and ( c ) A549-hsACE2 cells. The primary anti-vimentin polyclonal rabbit antibody that targets the phosphorylation site of SER56 on the N-terminus blocks uptake in A549-hsACE2 cells but not HEK 293T-hsACE2 cells. Denotations: *, P ≤ 0.05; **, P < 0.01; ***, P < 0.001; NS, P > 0.05.

Article Snippet: Briefly, for AFM evaluation of pseudoviruses in control (non-vimentin addition) and samples preincubated with human recombinant vimentin, a 10 μl drop of pseudoviruses or pseudoviruses/vimentin was applied on cleaved mica (SPI Supplies, West Chester, PA) and analyzed in a dried environment.

Techniques: Immunofluorescence, Recombinant

Journal: bioRxiv

Article Title: Extracellular vimentin as a target against SARS-CoV-2 host cell invasion

doi: 10.1101/2021.01.08.425793

Figure Lengend Snippet: List of anti-vimentin antibodies

Article Snippet: Briefly, for AFM evaluation of pseudoviruses in control (non-vimentin addition) and samples preincubated with human recombinant vimentin, a 10 μl drop of pseudoviruses or pseudoviruses/vimentin was applied on cleaved mica (SPI Supplies, West Chester, PA) and analyzed in a dried environment.

Techniques: Binding Assay, Blocking Assay, Produced, Recombinant, Synthesized, Derivative Assay