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mouse vimentin enzyme-linked immunosorbent assays (elisa) kit  (MyBiosource Biotechnology)

 
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    MyBiosource Biotechnology mouse vimentin enzyme-linked immunosorbent assays (elisa) kit
    Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), <t>vimentin,</t> collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.
    Mouse Vimentin Enzyme Linked Immunosorbent Assays (Elisa) Kit, supplied by MyBiosource Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+vimentin+enzyme-linked+immunosorbent+assays+%28elisa%29+kit/pmc05538875-95-13-20?v=MyBiosource+Biotechnology
    Average 90 stars, based on 1 article reviews
    mouse vimentin enzyme-linked immunosorbent assays (elisa) kit - by Bioz Stars, 2026-08
    90/100 stars

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    1) Product Images from "Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin"

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00087.2017

    Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), vimentin, collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.
    Figure Legend Snippet: Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), vimentin, collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.

    Techniques Used: Saline, Staining, Control, Sircol Collagen Assay

    Cd induces phosphorylation of AKT and cdc2 as well as vimentin phosphorylation at Ser39 and Ser55. A and B: after 24 h of serum starvation, primary lung fibroblasts were treated with CdCl2 (20 μM) for the indicated times. Immunoblotting were performed using antibodies shown above. The results shown in A and B are representative of those from 3 independent experiments. Arrows represent specific band.
    Figure Legend Snippet: Cd induces phosphorylation of AKT and cdc2 as well as vimentin phosphorylation at Ser39 and Ser55. A and B: after 24 h of serum starvation, primary lung fibroblasts were treated with CdCl2 (20 μM) for the indicated times. Immunoblotting were performed using antibodies shown above. The results shown in A and B are representative of those from 3 independent experiments. Arrows represent specific band.

    Techniques Used: Phospho-proteomics, Western Blot

    Both AKT and cdc2 inhibitors inhibit Cd-induced vimentin phosphorylation at Ser39 and Ser55, α-SMA activation, ECM accumulation, and collagen secretion. A; after 24 h of serum starvation, primary lung fibroblasts (n = 5) were pretreated with the MEK 1 inhibitor PD 985059 (PD; 20 μM), the PKC inhibitor G06983 (G0; 250 nM), the AKT inhibitor LY294002 (LY; 20 μM), the cdc2 inhibitor Roscovitine (RO; 10 μM) only, or combination of AKT and cdc2 inhibitors (LY + RO) 1 h before 20 μM CdCl2 treatment for 3 h and then allowed to recover for 48 h. The soluble collagen levels were evaluated by Sircol collagen assay. Horizontal bars indicate the mean values. **P < 0.01 vs. control. Cells were also treated with TGF-β (2 ng/ml) for 48 h as a positive control. B and C: cells were pretreated with inhibitor shown as above before 20 μM CdCl2 treatment for 2 h (B) or 3 h and then allowed to recover for 48 h (C), followed by immunoblot analysis for indicated antibodies. The results shown in A–C are representative of those from 3 independent experiments.
    Figure Legend Snippet: Both AKT and cdc2 inhibitors inhibit Cd-induced vimentin phosphorylation at Ser39 and Ser55, α-SMA activation, ECM accumulation, and collagen secretion. A; after 24 h of serum starvation, primary lung fibroblasts (n = 5) were pretreated with the MEK 1 inhibitor PD 985059 (PD; 20 μM), the PKC inhibitor G06983 (G0; 250 nM), the AKT inhibitor LY294002 (LY; 20 μM), the cdc2 inhibitor Roscovitine (RO; 10 μM) only, or combination of AKT and cdc2 inhibitors (LY + RO) 1 h before 20 μM CdCl2 treatment for 3 h and then allowed to recover for 48 h. The soluble collagen levels were evaluated by Sircol collagen assay. Horizontal bars indicate the mean values. **P < 0.01 vs. control. Cells were also treated with TGF-β (2 ng/ml) for 48 h as a positive control. B and C: cells were pretreated with inhibitor shown as above before 20 μM CdCl2 treatment for 2 h (B) or 3 h and then allowed to recover for 48 h (C), followed by immunoblot analysis for indicated antibodies. The results shown in A–C are representative of those from 3 independent experiments.

    Techniques Used: Phospho-proteomics, Activation Assay, Sircol Collagen Assay, Control, Positive Control, Western Blot

    Cd-induced vimentin fragmentation and secretion can be inhibited by both AKT and cdc2 inhibitors. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 or 0.018 mg/kg) as shown in Fig. 1. Plasma and bronchoalveolar lavage (BAL) were collected at week 4 and the vimentin levels were examined by ELISA. Horizontal bars indicate the mean values. B: primary lung fibroblasts were incubated with or without CdCl2 (20 μM) for 3 h and then allowed to recover for the indicated periods of time, and the supernatants were collected, concentrated by ultrafiltration and equal aliquots were analyzed by Western blotting using an anti-vimentin antibody. C: cells were pretreated with the AKT inhibitor (LY; 20 μM), the cdc2 inhibitor (RO; 10 μM), or a combination of AKT and cdc2 inhibitors (LY + RO) for 1 h, followed by 20 μM of CdCl2 treatment for 3 h and then allowed to recover for 48 h. Supernatants was analyzed as above. *P < 0.05, **P < 0.01. Arrows represent positions for the indicated molecular weight. The data are representative of 3 independent experiments.
    Figure Legend Snippet: Cd-induced vimentin fragmentation and secretion can be inhibited by both AKT and cdc2 inhibitors. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 or 0.018 mg/kg) as shown in Fig. 1. Plasma and bronchoalveolar lavage (BAL) were collected at week 4 and the vimentin levels were examined by ELISA. Horizontal bars indicate the mean values. B: primary lung fibroblasts were incubated with or without CdCl2 (20 μM) for 3 h and then allowed to recover for the indicated periods of time, and the supernatants were collected, concentrated by ultrafiltration and equal aliquots were analyzed by Western blotting using an anti-vimentin antibody. C: cells were pretreated with the AKT inhibitor (LY; 20 μM), the cdc2 inhibitor (RO; 10 μM), or a combination of AKT and cdc2 inhibitors (LY + RO) for 1 h, followed by 20 μM of CdCl2 treatment for 3 h and then allowed to recover for 48 h. Supernatants was analyzed as above. *P < 0.05, **P < 0.01. Arrows represent positions for the indicated molecular weight. The data are representative of 3 independent experiments.

    Techniques Used: Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Molecular Weight

    Vimentin is required for Cd-induced peribronchiolar fibrosis. A and B: IMR-90 cells were transfected with vimentin siRNA or NT-siRNA and followed by 20 μM of CdCl2 treatment for 2 h (A) or 3 h and then allowed to recover for 48 h (B). Western blots were performed using antibodies shown above. C: the soluble collagen levels were evaluated by Sircol collagen assay (n = 5). **P < 0.01. D: wild-type (WT) and Vimentin−/− mice (n = 5 of each group) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with collagen-1 or picro-sirius red. Bar = 100 μm. Representative photomicrographs from 5 animals on week 4 are shown. E: 3,3′-diaminobenzidine staining density was quantified and the average positive area density was calculated from 10 random areas. Densities are denoted in arbitrary units. **P < 0.01. F: WT and Vimentin−/− mice (n = 5 of each group) were treated as above and airway resistance following methacholine challenge was measured. ##P < 0.01 vs. WT Cd mice with methacholine at 20 or 40 mg/ml. Horizontal bars indicate the mean values.
    Figure Legend Snippet: Vimentin is required for Cd-induced peribronchiolar fibrosis. A and B: IMR-90 cells were transfected with vimentin siRNA or NT-siRNA and followed by 20 μM of CdCl2 treatment for 2 h (A) or 3 h and then allowed to recover for 48 h (B). Western blots were performed using antibodies shown above. C: the soluble collagen levels were evaluated by Sircol collagen assay (n = 5). **P < 0.01. D: wild-type (WT) and Vimentin−/− mice (n = 5 of each group) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with collagen-1 or picro-sirius red. Bar = 100 μm. Representative photomicrographs from 5 animals on week 4 are shown. E: 3,3′-diaminobenzidine staining density was quantified and the average positive area density was calculated from 10 random areas. Densities are denoted in arbitrary units. **P < 0.01. F: WT and Vimentin−/− mice (n = 5 of each group) were treated as above and airway resistance following methacholine challenge was measured. ##P < 0.01 vs. WT Cd mice with methacholine at 20 or 40 mg/ml. Horizontal bars indicate the mean values.

    Techniques Used: Transfection, Western Blot, Sircol Collagen Assay, Saline, Staining

    Cd induces nuclear localization and association of Yes-associated protein 1 (YAP1) and Smad2/3. A: primary lung fibroblasts were incubated with or without CdCl2 (10 or 20 μM) or TGF-β (2 ng/ml) for 2 h and followed by immunoblot analysis with anti-P-Smad2/3 or anti-Smad2/3. B: cells were subjected to subcellular fractionation to obtain nuclear and cytoplasm fractions. Equivalent protein amounts of each fraction were separated by SDS-PAGE, and immunoblotted with anti-Smad2/3, YAP1, 14-3-3, or anti-vimentin. Tubulin, lamin, and β-actin were used as loading controls. C: cells were pretreated with AKT inhibitor (LY; 20 μM) and cdc2 inhibitor (RO; 10 μM) or phosphatase inhibitor [calf intestinal phosphatase (CIP); 10 units/ml] for 1 h and then stimulated with CdCl2 for 2 h, lysed, and subjected to YAP1 or vimentin immunoprecipitation followed by Smad2/3, 14-3-3, vimentin, or YAP1 Western analysis. The densities of protein bands were determined by densitometry and the data represent a 1-fold increase from the control density. **P < 0.01. Horizontal bars indicate the mean values. The results shown in A–C are representative of those from 3 independent experiments.
    Figure Legend Snippet: Cd induces nuclear localization and association of Yes-associated protein 1 (YAP1) and Smad2/3. A: primary lung fibroblasts were incubated with or without CdCl2 (10 or 20 μM) or TGF-β (2 ng/ml) for 2 h and followed by immunoblot analysis with anti-P-Smad2/3 or anti-Smad2/3. B: cells were subjected to subcellular fractionation to obtain nuclear and cytoplasm fractions. Equivalent protein amounts of each fraction were separated by SDS-PAGE, and immunoblotted with anti-Smad2/3, YAP1, 14-3-3, or anti-vimentin. Tubulin, lamin, and β-actin were used as loading controls. C: cells were pretreated with AKT inhibitor (LY; 20 μM) and cdc2 inhibitor (RO; 10 μM) or phosphatase inhibitor [calf intestinal phosphatase (CIP); 10 units/ml] for 1 h and then stimulated with CdCl2 for 2 h, lysed, and subjected to YAP1 or vimentin immunoprecipitation followed by Smad2/3, 14-3-3, vimentin, or YAP1 Western analysis. The densities of protein bands were determined by densitometry and the data represent a 1-fold increase from the control density. **P < 0.01. Horizontal bars indicate the mean values. The results shown in A–C are representative of those from 3 independent experiments.

    Techniques Used: Incubation, Western Blot, Fractionation, SDS Page, Immunoprecipitation, Control

    Proposed model for the role of vimentin phosphorylation and YAP1 in Cd-induced peribronchiolar fibrosis, airway remodeling, and subsequent COPD. Prolonged exposure to Cd results in fibrosis around the airways. Cd-induced phosphorylation of AKT and cdc2 kinase is required for the vimentin phosphorylation at Ser39 and Ser55, respectively. Such phosphorylated vimentin will bind 14-3-3 and facilitate the nuclear translocation of YAP1 and Smad2/3 and complex formation of YAP1 and Smad2/3 in the nucleus, which activates myofibroblast and induces ECM protein deposition.
    Figure Legend Snippet: Proposed model for the role of vimentin phosphorylation and YAP1 in Cd-induced peribronchiolar fibrosis, airway remodeling, and subsequent COPD. Prolonged exposure to Cd results in fibrosis around the airways. Cd-induced phosphorylation of AKT and cdc2 kinase is required for the vimentin phosphorylation at Ser39 and Ser55, respectively. Such phosphorylated vimentin will bind 14-3-3 and facilitate the nuclear translocation of YAP1 and Smad2/3 and complex formation of YAP1 and Smad2/3 in the nucleus, which activates myofibroblast and induces ECM protein deposition.

    Techniques Used: Phospho-proteomics, Translocation Assay



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    mouse vimentin enzyme-linked immunosorbent assays (elisa) kit  (MyBiosource Biotechnology)
    90
    MyBiosource Biotechnology mouse vimentin enzyme-linked immunosorbent assays (elisa) kit
    Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), <t>vimentin,</t> collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.
    Mouse Vimentin Enzyme Linked Immunosorbent Assays (Elisa) Kit, supplied by MyBiosource Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+vimentin+enzyme-linked+immunosorbent+assays+%28elisa%29+kit/pmc05538875-95-13-20?v=MyBiosource+Biotechnology
    Average 90 stars, based on 1 article reviews
    mouse vimentin enzyme-linked immunosorbent assays (elisa) kit - by Bioz Stars, 2026-08
    90/100 stars
    		  Buy from Supplier

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    Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), vimentin, collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Cadmium (Cd) induces peribronchiolar fibrosis. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with hematoxylin-eosin, α-smooth muscle actin (α-SMA), vimentin, collagen-1, or picro-sirius red. Representative photomicrographs from 5 animals on week 4 are shown, and peribronchiolar fibrosis is indicated by arrows. Bar = 100 μm. B: the peribronchiolar subepithelial layer in mice (n = 5) was measured and comparable to the saline control. Each symbol represents an individual subjects, and horizontal bars indicate the mean values. C: respiratory mechanics demonstrating increased airway resistance following methacholine challenge. ##P < 0.01 vs. saline mice with methacholine at 20 or 40 mg/ml. D: mice were treated as shown in A, and collagen content in the right lung at different time points was assessed using Sircol collagen assay. **P < 0.01 vs. saline control.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Saline, Staining, Control, Sircol Collagen Assay

    Cd induces phosphorylation of AKT and cdc2 as well as vimentin phosphorylation at Ser39 and Ser55. A and B: after 24 h of serum starvation, primary lung fibroblasts were treated with CdCl2 (20 μM) for the indicated times. Immunoblotting were performed using antibodies shown above. The results shown in A and B are representative of those from 3 independent experiments. Arrows represent specific band.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Cd induces phosphorylation of AKT and cdc2 as well as vimentin phosphorylation at Ser39 and Ser55. A and B: after 24 h of serum starvation, primary lung fibroblasts were treated with CdCl2 (20 μM) for the indicated times. Immunoblotting were performed using antibodies shown above. The results shown in A and B are representative of those from 3 independent experiments. Arrows represent specific band.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Phospho-proteomics, Western Blot

    Both AKT and cdc2 inhibitors inhibit Cd-induced vimentin phosphorylation at Ser39 and Ser55, α-SMA activation, ECM accumulation, and collagen secretion. A; after 24 h of serum starvation, primary lung fibroblasts (n = 5) were pretreated with the MEK 1 inhibitor PD 985059 (PD; 20 μM), the PKC inhibitor G06983 (G0; 250 nM), the AKT inhibitor LY294002 (LY; 20 μM), the cdc2 inhibitor Roscovitine (RO; 10 μM) only, or combination of AKT and cdc2 inhibitors (LY + RO) 1 h before 20 μM CdCl2 treatment for 3 h and then allowed to recover for 48 h. The soluble collagen levels were evaluated by Sircol collagen assay. Horizontal bars indicate the mean values. **P < 0.01 vs. control. Cells were also treated with TGF-β (2 ng/ml) for 48 h as a positive control. B and C: cells were pretreated with inhibitor shown as above before 20 μM CdCl2 treatment for 2 h (B) or 3 h and then allowed to recover for 48 h (C), followed by immunoblot analysis for indicated antibodies. The results shown in A–C are representative of those from 3 independent experiments.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Both AKT and cdc2 inhibitors inhibit Cd-induced vimentin phosphorylation at Ser39 and Ser55, α-SMA activation, ECM accumulation, and collagen secretion. A; after 24 h of serum starvation, primary lung fibroblasts (n = 5) were pretreated with the MEK 1 inhibitor PD 985059 (PD; 20 μM), the PKC inhibitor G06983 (G0; 250 nM), the AKT inhibitor LY294002 (LY; 20 μM), the cdc2 inhibitor Roscovitine (RO; 10 μM) only, or combination of AKT and cdc2 inhibitors (LY + RO) 1 h before 20 μM CdCl2 treatment for 3 h and then allowed to recover for 48 h. The soluble collagen levels were evaluated by Sircol collagen assay. Horizontal bars indicate the mean values. **P < 0.01 vs. control. Cells were also treated with TGF-β (2 ng/ml) for 48 h as a positive control. B and C: cells were pretreated with inhibitor shown as above before 20 μM CdCl2 treatment for 2 h (B) or 3 h and then allowed to recover for 48 h (C), followed by immunoblot analysis for indicated antibodies. The results shown in A–C are representative of those from 3 independent experiments.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Phospho-proteomics, Activation Assay, Sircol Collagen Assay, Control, Positive Control, Western Blot

    Cd-induced vimentin fragmentation and secretion can be inhibited by both AKT and cdc2 inhibitors. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 or 0.018 mg/kg) as shown in Fig. 1. Plasma and bronchoalveolar lavage (BAL) were collected at week 4 and the vimentin levels were examined by ELISA. Horizontal bars indicate the mean values. B: primary lung fibroblasts were incubated with or without CdCl2 (20 μM) for 3 h and then allowed to recover for the indicated periods of time, and the supernatants were collected, concentrated by ultrafiltration and equal aliquots were analyzed by Western blotting using an anti-vimentin antibody. C: cells were pretreated with the AKT inhibitor (LY; 20 μM), the cdc2 inhibitor (RO; 10 μM), or a combination of AKT and cdc2 inhibitors (LY + RO) for 1 h, followed by 20 μM of CdCl2 treatment for 3 h and then allowed to recover for 48 h. Supernatants was analyzed as above. *P < 0.05, **P < 0.01. Arrows represent positions for the indicated molecular weight. The data are representative of 3 independent experiments.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Cd-induced vimentin fragmentation and secretion can be inhibited by both AKT and cdc2 inhibitors. A: mice (n = 5) were treated intratracheally with saline or CdCl2 (0.009 or 0.018 mg/kg) as shown in Fig. 1. Plasma and bronchoalveolar lavage (BAL) were collected at week 4 and the vimentin levels were examined by ELISA. Horizontal bars indicate the mean values. B: primary lung fibroblasts were incubated with or without CdCl2 (20 μM) for 3 h and then allowed to recover for the indicated periods of time, and the supernatants were collected, concentrated by ultrafiltration and equal aliquots were analyzed by Western blotting using an anti-vimentin antibody. C: cells were pretreated with the AKT inhibitor (LY; 20 μM), the cdc2 inhibitor (RO; 10 μM), or a combination of AKT and cdc2 inhibitors (LY + RO) for 1 h, followed by 20 μM of CdCl2 treatment for 3 h and then allowed to recover for 48 h. Supernatants was analyzed as above. *P < 0.05, **P < 0.01. Arrows represent positions for the indicated molecular weight. The data are representative of 3 independent experiments.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Molecular Weight

    Vimentin is required for Cd-induced peribronchiolar fibrosis. A and B: IMR-90 cells were transfected with vimentin siRNA or NT-siRNA and followed by 20 μM of CdCl2 treatment for 2 h (A) or 3 h and then allowed to recover for 48 h (B). Western blots were performed using antibodies shown above. C: the soluble collagen levels were evaluated by Sircol collagen assay (n = 5). **P < 0.01. D: wild-type (WT) and Vimentin−/− mice (n = 5 of each group) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with collagen-1 or picro-sirius red. Bar = 100 μm. Representative photomicrographs from 5 animals on week 4 are shown. E: 3,3′-diaminobenzidine staining density was quantified and the average positive area density was calculated from 10 random areas. Densities are denoted in arbitrary units. **P < 0.01. F: WT and Vimentin−/− mice (n = 5 of each group) were treated as above and airway resistance following methacholine challenge was measured. ##P < 0.01 vs. WT Cd mice with methacholine at 20 or 40 mg/ml. Horizontal bars indicate the mean values.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Vimentin is required for Cd-induced peribronchiolar fibrosis. A and B: IMR-90 cells were transfected with vimentin siRNA or NT-siRNA and followed by 20 μM of CdCl2 treatment for 2 h (A) or 3 h and then allowed to recover for 48 h (B). Western blots were performed using antibodies shown above. C: the soluble collagen levels were evaluated by Sircol collagen assay (n = 5). **P < 0.01. D: wild-type (WT) and Vimentin−/− mice (n = 5 of each group) were treated intratracheally with saline or CdCl2 (0.009 mg/kg body wt) every other day for 8 wk. Cryosections from weeks 1, 2, 4, and 8 mice lungs were stained with collagen-1 or picro-sirius red. Bar = 100 μm. Representative photomicrographs from 5 animals on week 4 are shown. E: 3,3′-diaminobenzidine staining density was quantified and the average positive area density was calculated from 10 random areas. Densities are denoted in arbitrary units. **P < 0.01. F: WT and Vimentin−/− mice (n = 5 of each group) were treated as above and airway resistance following methacholine challenge was measured. ##P < 0.01 vs. WT Cd mice with methacholine at 20 or 40 mg/ml. Horizontal bars indicate the mean values.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Transfection, Western Blot, Sircol Collagen Assay, Saline, Staining

    Cd induces nuclear localization and association of Yes-associated protein 1 (YAP1) and Smad2/3. A: primary lung fibroblasts were incubated with or without CdCl2 (10 or 20 μM) or TGF-β (2 ng/ml) for 2 h and followed by immunoblot analysis with anti-P-Smad2/3 or anti-Smad2/3. B: cells were subjected to subcellular fractionation to obtain nuclear and cytoplasm fractions. Equivalent protein amounts of each fraction were separated by SDS-PAGE, and immunoblotted with anti-Smad2/3, YAP1, 14-3-3, or anti-vimentin. Tubulin, lamin, and β-actin were used as loading controls. C: cells were pretreated with AKT inhibitor (LY; 20 μM) and cdc2 inhibitor (RO; 10 μM) or phosphatase inhibitor [calf intestinal phosphatase (CIP); 10 units/ml] for 1 h and then stimulated with CdCl2 for 2 h, lysed, and subjected to YAP1 or vimentin immunoprecipitation followed by Smad2/3, 14-3-3, vimentin, or YAP1 Western analysis. The densities of protein bands were determined by densitometry and the data represent a 1-fold increase from the control density. **P < 0.01. Horizontal bars indicate the mean values. The results shown in A–C are representative of those from 3 independent experiments.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Cd induces nuclear localization and association of Yes-associated protein 1 (YAP1) and Smad2/3. A: primary lung fibroblasts were incubated with or without CdCl2 (10 or 20 μM) or TGF-β (2 ng/ml) for 2 h and followed by immunoblot analysis with anti-P-Smad2/3 or anti-Smad2/3. B: cells were subjected to subcellular fractionation to obtain nuclear and cytoplasm fractions. Equivalent protein amounts of each fraction were separated by SDS-PAGE, and immunoblotted with anti-Smad2/3, YAP1, 14-3-3, or anti-vimentin. Tubulin, lamin, and β-actin were used as loading controls. C: cells were pretreated with AKT inhibitor (LY; 20 μM) and cdc2 inhibitor (RO; 10 μM) or phosphatase inhibitor [calf intestinal phosphatase (CIP); 10 units/ml] for 1 h and then stimulated with CdCl2 for 2 h, lysed, and subjected to YAP1 or vimentin immunoprecipitation followed by Smad2/3, 14-3-3, vimentin, or YAP1 Western analysis. The densities of protein bands were determined by densitometry and the data represent a 1-fold increase from the control density. **P < 0.01. Horizontal bars indicate the mean values. The results shown in A–C are representative of those from 3 independent experiments.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Incubation, Western Blot, Fractionation, SDS Page, Immunoprecipitation, Control

    Proposed model for the role of vimentin phosphorylation and YAP1 in Cd-induced peribronchiolar fibrosis, airway remodeling, and subsequent COPD. Prolonged exposure to Cd results in fibrosis around the airways. Cd-induced phosphorylation of AKT and cdc2 kinase is required for the vimentin phosphorylation at Ser39 and Ser55, respectively. Such phosphorylated vimentin will bind 14-3-3 and facilitate the nuclear translocation of YAP1 and Smad2/3 and complex formation of YAP1 and Smad2/3 in the nucleus, which activates myofibroblast and induces ECM protein deposition.

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin

    doi: 10.1152/ajplung.00087.2017

    Figure Lengend Snippet: Proposed model for the role of vimentin phosphorylation and YAP1 in Cd-induced peribronchiolar fibrosis, airway remodeling, and subsequent COPD. Prolonged exposure to Cd results in fibrosis around the airways. Cd-induced phosphorylation of AKT and cdc2 kinase is required for the vimentin phosphorylation at Ser39 and Ser55, respectively. Such phosphorylated vimentin will bind 14-3-3 and facilitate the nuclear translocation of YAP1 and Smad2/3 and complex formation of YAP1 and Smad2/3 in the nucleus, which activates myofibroblast and induces ECM protein deposition.

    Article Snippet: Vimentin concentrations in mice plasma and BAL were quantified using a commercially available mouse vimentin enzyme-linked immunosorbent assays (ELISA) kit (MyBioSource, San Diego, CA) according to the manufacturer’s recommendation.

    Techniques: Phospho-proteomics, Translocation Assay