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  • 95
    Chem Impex International emission 39
    Emission 39, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/emission 39/product/Chem Impex International
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    94
    Bio X Cell anti mouse cd49d mab
    MZB cell immunodepletion decreases anti-H2 b antibody production in alloimmunized mice. (A) Representative dot plots of the gating strategy used for quantification of splenic MZB cells (B220 + IgM + CD23 − CD21 ++ ). BALB/C recipient mice were treated with anti-mouse CD11a and anti-mouse <t>CD49d</t> antibodies (lower panels) or their respective isotype-matched controls (IgG1 and IgG2a, upper panels). (B) Quantification of the percentage of splenic MZB cells among the CD45 + cells in mice treated with anti-CD11a/CD49d antibodies or their isotype controls (n = 5). (C) Anti-H2 b IgG antibody production was evaluated weekly by flow cytometry in immunodepleted MZB cell (blue bars) and mice that received isotype control (red bars) BALB/C mice. Results are presented as the mean fluorescence intensity (MFI ± SEM) of Alexa-488 GaM IgG antibodies bound to H2 b platelets (∗ P < .05; ∗∗ P < .01; 1-way ANOVA; n = 18). (D) The kinetics of elimination of transfused platelets were determined as the percentage of Oregon green-positive platelets among 10 000 RAM.1 + platelets in MZB cell depleted (blue line) and control mice (red line) at each time point (± SEM; ∗∗ P < .01; ∗∗∗ P < .001; 2-way ANOVA; n = 8-9). ANOVA, analysis of variance; SEM, standard error of the mean.
    Anti Mouse Cd49d Mab, supplied by Bio X Cell, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Proteintech α cavin 2
    Identification <t>Cavin-2</t> as a novel angiogenesis regulator. A , confocal microscopy images of the Tg( fli1a :eGFP) y1 embryos microinjected with Cavin-2 gene-specific morpholinos ( sdpra-mo1 and sdpra-mo2 ) or control morpholinos. Top row represents differential interference contrast ( DIC ) images, middle row represents EGFP signals from Tg( fli1a :eGFP) y1 , and the bottom row represents the zoomed inset from the images from middle row . The EGFP panels shown are from the morpholino screen represented in B and represented here to show that there no major phenotypic differences between the morphants and control, and for a detailed view on the defects on intersomitic vessels of Cavin-2 morphants with respect to control morphants. Scale , 1 mm. B , immunoblots of <t>α-Cavin-2</t> and α–β-actin antibodies on the WCL of HAEC, HUVEC, and HPMEC cells shows an higher expression of Cavin-2. β-Actin serves as a loading control. C , immunofluorescence staining of CD31 and Cavin-2 in P17 mouse retina after OIR. Cavin-2 expression is enriched at the neovascular tufts ( white arrows ), whereas low constitutive expression is associated with the surrounding non-aberrant vessels ( yellow arrow ). Scale , 20 μm. D , an increased Cavin-2 mRNA expression is associated with increased vessel density in developing mouse retina. One-way analysis of variance followed by Tukey's multiple comparisons test. Data are expressed as mean ± S.D. ( n ≥ 3 animals); *, p < 0.05. E , the immunofluorescence staining of Cavin-2 indicates a low-constitutive and homogenous expression in retinal vasculature of P7 mouse pups and low levels in mature retinal vasculature at P21. Scale , 20 μm.
    α Cavin 2, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Proteintech presenilin 1 ps1
    Identification <t>Cavin-2</t> as a novel angiogenesis regulator. A , confocal microscopy images of the Tg( fli1a :eGFP) y1 embryos microinjected with Cavin-2 gene-specific morpholinos ( sdpra-mo1 and sdpra-mo2 ) or control morpholinos. Top row represents differential interference contrast ( DIC ) images, middle row represents EGFP signals from Tg( fli1a :eGFP) y1 , and the bottom row represents the zoomed inset from the images from middle row . The EGFP panels shown are from the morpholino screen represented in B and represented here to show that there no major phenotypic differences between the morphants and control, and for a detailed view on the defects on intersomitic vessels of Cavin-2 morphants with respect to control morphants. Scale , 1 mm. B , immunoblots of <t>α-Cavin-2</t> and α–β-actin antibodies on the WCL of HAEC, HUVEC, and HPMEC cells shows an higher expression of Cavin-2. β-Actin serves as a loading control. C , immunofluorescence staining of CD31 and Cavin-2 in P17 mouse retina after OIR. Cavin-2 expression is enriched at the neovascular tufts ( white arrows ), whereas low constitutive expression is associated with the surrounding non-aberrant vessels ( yellow arrow ). Scale , 20 μm. D , an increased Cavin-2 mRNA expression is associated with increased vessel density in developing mouse retina. One-way analysis of variance followed by Tukey's multiple comparisons test. Data are expressed as mean ± S.D. ( n ≥ 3 animals); *, p < 0.05. E , the immunofluorescence staining of Cavin-2 indicates a low-constitutive and homogenous expression in retinal vasculature of P7 mouse pups and low levels in mature retinal vasculature at P21. Scale , 20 μm.
    Presenilin 1 Ps1, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Proteintech arhgap10
    Identification <t>Cavin-2</t> as a novel angiogenesis regulator. A , confocal microscopy images of the Tg( fli1a :eGFP) y1 embryos microinjected with Cavin-2 gene-specific morpholinos ( sdpra-mo1 and sdpra-mo2 ) or control morpholinos. Top row represents differential interference contrast ( DIC ) images, middle row represents EGFP signals from Tg( fli1a :eGFP) y1 , and the bottom row represents the zoomed inset from the images from middle row . The EGFP panels shown are from the morpholino screen represented in B and represented here to show that there no major phenotypic differences between the morphants and control, and for a detailed view on the defects on intersomitic vessels of Cavin-2 morphants with respect to control morphants. Scale , 1 mm. B , immunoblots of <t>α-Cavin-2</t> and α–β-actin antibodies on the WCL of HAEC, HUVEC, and HPMEC cells shows an higher expression of Cavin-2. β-Actin serves as a loading control. C , immunofluorescence staining of CD31 and Cavin-2 in P17 mouse retina after OIR. Cavin-2 expression is enriched at the neovascular tufts ( white arrows ), whereas low constitutive expression is associated with the surrounding non-aberrant vessels ( yellow arrow ). Scale , 20 μm. D , an increased Cavin-2 mRNA expression is associated with increased vessel density in developing mouse retina. One-way analysis of variance followed by Tukey's multiple comparisons test. Data are expressed as mean ± S.D. ( n ≥ 3 animals); *, p < 0.05. E , the immunofluorescence staining of Cavin-2 indicates a low-constitutive and homogenous expression in retinal vasculature of P7 mouse pups and low levels in mature retinal vasculature at P21. Scale , 20 μm.
    Arhgap10, supplied by Proteintech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    MZB cell immunodepletion decreases anti-H2 b antibody production in alloimmunized mice. (A) Representative dot plots of the gating strategy used for quantification of splenic MZB cells (B220 + IgM + CD23 − CD21 ++ ). BALB/C recipient mice were treated with anti-mouse CD11a and anti-mouse CD49d antibodies (lower panels) or their respective isotype-matched controls (IgG1 and IgG2a, upper panels). (B) Quantification of the percentage of splenic MZB cells among the CD45 + cells in mice treated with anti-CD11a/CD49d antibodies or their isotype controls (n = 5). (C) Anti-H2 b IgG antibody production was evaluated weekly by flow cytometry in immunodepleted MZB cell (blue bars) and mice that received isotype control (red bars) BALB/C mice. Results are presented as the mean fluorescence intensity (MFI ± SEM) of Alexa-488 GaM IgG antibodies bound to H2 b platelets (∗ P < .05; ∗∗ P < .01; 1-way ANOVA; n = 18). (D) The kinetics of elimination of transfused platelets were determined as the percentage of Oregon green-positive platelets among 10 000 RAM.1 + platelets in MZB cell depleted (blue line) and control mice (red line) at each time point (± SEM; ∗∗ P < .01; ∗∗∗ P < .001; 2-way ANOVA; n = 8-9). ANOVA, analysis of variance; SEM, standard error of the mean.

    Journal: Blood Advances

    Article Title: Marginal zone B cells are responsible for the production of alloantibodies following platelet transfusion in mice

    doi: 10.1182/bloodadvances.2022008411

    Figure Lengend Snippet: MZB cell immunodepletion decreases anti-H2 b antibody production in alloimmunized mice. (A) Representative dot plots of the gating strategy used for quantification of splenic MZB cells (B220 + IgM + CD23 − CD21 ++ ). BALB/C recipient mice were treated with anti-mouse CD11a and anti-mouse CD49d antibodies (lower panels) or their respective isotype-matched controls (IgG1 and IgG2a, upper panels). (B) Quantification of the percentage of splenic MZB cells among the CD45 + cells in mice treated with anti-CD11a/CD49d antibodies or their isotype controls (n = 5). (C) Anti-H2 b IgG antibody production was evaluated weekly by flow cytometry in immunodepleted MZB cell (blue bars) and mice that received isotype control (red bars) BALB/C mice. Results are presented as the mean fluorescence intensity (MFI ± SEM) of Alexa-488 GaM IgG antibodies bound to H2 b platelets (∗ P < .05; ∗∗ P < .01; 1-way ANOVA; n = 18). (D) The kinetics of elimination of transfused platelets were determined as the percentage of Oregon green-positive platelets among 10 000 RAM.1 + platelets in MZB cell depleted (blue line) and control mice (red line) at each time point (± SEM; ∗∗ P < .01; ∗∗∗ P < .001; 2-way ANOVA; n = 8-9). ANOVA, analysis of variance; SEM, standard error of the mean.

    Article Snippet: MZB cells were depleted 48 hours before platelet transfusion by intraperitoneal injection of an anti-mouse CD11a mAb (100 μg, clone M17/4; Bioxcell, Lebanon, NH) and an anti-mouse CD49d mAb (100 μg, clone PS/2; Bioxcell) or isotype-matched control antibodies (rat immunoglobulin G2a (IgG2a), clone 2A3, and rat IgG2b, clone LTF-2; Bioxcell) diluted in phosphate buffered saline.

    Techniques: Flow Cytometry, Fluorescence

    Identification Cavin-2 as a novel angiogenesis regulator. A , confocal microscopy images of the Tg( fli1a :eGFP) y1 embryos microinjected with Cavin-2 gene-specific morpholinos ( sdpra-mo1 and sdpra-mo2 ) or control morpholinos. Top row represents differential interference contrast ( DIC ) images, middle row represents EGFP signals from Tg( fli1a :eGFP) y1 , and the bottom row represents the zoomed inset from the images from middle row . The EGFP panels shown are from the morpholino screen represented in B and represented here to show that there no major phenotypic differences between the morphants and control, and for a detailed view on the defects on intersomitic vessels of Cavin-2 morphants with respect to control morphants. Scale , 1 mm. B , immunoblots of α-Cavin-2 and α–β-actin antibodies on the WCL of HAEC, HUVEC, and HPMEC cells shows an higher expression of Cavin-2. β-Actin serves as a loading control. C , immunofluorescence staining of CD31 and Cavin-2 in P17 mouse retina after OIR. Cavin-2 expression is enriched at the neovascular tufts ( white arrows ), whereas low constitutive expression is associated with the surrounding non-aberrant vessels ( yellow arrow ). Scale , 20 μm. D , an increased Cavin-2 mRNA expression is associated with increased vessel density in developing mouse retina. One-way analysis of variance followed by Tukey's multiple comparisons test. Data are expressed as mean ± S.D. ( n ≥ 3 animals); *, p < 0.05. E , the immunofluorescence staining of Cavin-2 indicates a low-constitutive and homogenous expression in retinal vasculature of P7 mouse pups and low levels in mature retinal vasculature at P21. Scale , 20 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Identification Cavin-2 as a novel angiogenesis regulator. A , confocal microscopy images of the Tg( fli1a :eGFP) y1 embryos microinjected with Cavin-2 gene-specific morpholinos ( sdpra-mo1 and sdpra-mo2 ) or control morpholinos. Top row represents differential interference contrast ( DIC ) images, middle row represents EGFP signals from Tg( fli1a :eGFP) y1 , and the bottom row represents the zoomed inset from the images from middle row . The EGFP panels shown are from the morpholino screen represented in B and represented here to show that there no major phenotypic differences between the morphants and control, and for a detailed view on the defects on intersomitic vessels of Cavin-2 morphants with respect to control morphants. Scale , 1 mm. B , immunoblots of α-Cavin-2 and α–β-actin antibodies on the WCL of HAEC, HUVEC, and HPMEC cells shows an higher expression of Cavin-2. β-Actin serves as a loading control. C , immunofluorescence staining of CD31 and Cavin-2 in P17 mouse retina after OIR. Cavin-2 expression is enriched at the neovascular tufts ( white arrows ), whereas low constitutive expression is associated with the surrounding non-aberrant vessels ( yellow arrow ). Scale , 20 μm. D , an increased Cavin-2 mRNA expression is associated with increased vessel density in developing mouse retina. One-way analysis of variance followed by Tukey's multiple comparisons test. Data are expressed as mean ± S.D. ( n ≥ 3 animals); *, p < 0.05. E , the immunofluorescence staining of Cavin-2 indicates a low-constitutive and homogenous expression in retinal vasculature of P7 mouse pups and low levels in mature retinal vasculature at P21. Scale , 20 μm.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

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

    Cavin-2 is required for cell proliferation, migration, and invasion in HUVEC cells. A, Western blot analysis of the WCLs from HUVECs after knockdown of non-targeting control ( siControl ) or cavin-2 ( siCavin-2 ) using siRNAs shows a severe loss of Cavin-2 expression. β-Actin serves as a loading control. B , siControl and siCavin-2 HUVECs were analyzed for cell proliferation at the mentioned time points using MTT assay showing a defective cell proliferation of siCavin-2 HUVECs. C , control and Cavin-2 knockdown HUVEC cells were analyzed for cell migration using a wound healing assay. The cells shown are pictured post-scratch at 0 and 16 h showing a defective cell migration after Cavin-2 loss. D , areas of the wound closure were quantified at 16 h after scratch using ImageJ. E , cell migration analyzed using control and Cavin-2 knockdown HUVECs using a transwell migration chamber (8-micron pore diameter) after being incubated for 12 h show a defective cell migration of sicavin-2 HUEVCs. F , quantification of the migrated crystal violet-stained endothelial cells after 12 h of migration using ImageJ. G , Matrigel invasion of control and Cavin-2 knockdown HUVECs after 48 h using a Boyden chamber assay signify that cavin-2 loss severely affects invasive potential of HUVECs. The cells were fixed in 4% paraformaldehyde and stained with crystal violet. H , quantification of the cells invasion through the Matrigel-coated filter was performed using ImageJ. Results in the graph presented are mean ± S.D. of three independent experiments. * indicates p < 0.05.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Cavin-2 is required for cell proliferation, migration, and invasion in HUVEC cells. A, Western blot analysis of the WCLs from HUVECs after knockdown of non-targeting control ( siControl ) or cavin-2 ( siCavin-2 ) using siRNAs shows a severe loss of Cavin-2 expression. β-Actin serves as a loading control. B , siControl and siCavin-2 HUVECs were analyzed for cell proliferation at the mentioned time points using MTT assay showing a defective cell proliferation of siCavin-2 HUVECs. C , control and Cavin-2 knockdown HUVEC cells were analyzed for cell migration using a wound healing assay. The cells shown are pictured post-scratch at 0 and 16 h showing a defective cell migration after Cavin-2 loss. D , areas of the wound closure were quantified at 16 h after scratch using ImageJ. E , cell migration analyzed using control and Cavin-2 knockdown HUVECs using a transwell migration chamber (8-micron pore diameter) after being incubated for 12 h show a defective cell migration of sicavin-2 HUEVCs. F , quantification of the migrated crystal violet-stained endothelial cells after 12 h of migration using ImageJ. G , Matrigel invasion of control and Cavin-2 knockdown HUVECs after 48 h using a Boyden chamber assay signify that cavin-2 loss severely affects invasive potential of HUVECs. The cells were fixed in 4% paraformaldehyde and stained with crystal violet. H , quantification of the cells invasion through the Matrigel-coated filter was performed using ImageJ. Results in the graph presented are mean ± S.D. of three independent experiments. * indicates p < 0.05.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: Migration, Western Blot, Expressing, MTT Assay, Wound Healing Assay, Incubation, Staining, Boyden Chamber Assay

    Loss of Cavin-2 inhibits angiogenesis in vitro in multiple endothelial cells. The Matrigel in vitro tube-formation assay was performed on control knockdown ( si-Control ) and Cavin-2 knockdown ( siCavin-2 ) in the following endothelial cells: A , HAEC. C , HUVEC. E , HPMEC. G , HRMVEC. Around 5000–7000 cells with endothelial cell growth media were plated on the Matrigel basement membrane matrix and allowed to form tube-like structures and recorded using a Nikon microscope. The graphs represent quantitation of mean ± S.D. of the number of branches and the total network length in in vitro tube-formation assay of: B , HAEC; D , HUVEC; F , HPMEC; and H , HRMVEC computed using ImageJ. * indicates p < 0.05.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Loss of Cavin-2 inhibits angiogenesis in vitro in multiple endothelial cells. The Matrigel in vitro tube-formation assay was performed on control knockdown ( si-Control ) and Cavin-2 knockdown ( siCavin-2 ) in the following endothelial cells: A , HAEC. C , HUVEC. E , HPMEC. G , HRMVEC. Around 5000–7000 cells with endothelial cell growth media were plated on the Matrigel basement membrane matrix and allowed to form tube-like structures and recorded using a Nikon microscope. The graphs represent quantitation of mean ± S.D. of the number of branches and the total network length in in vitro tube-formation assay of: B , HAEC; D , HUVEC; F , HPMEC; and H , HRMVEC computed using ImageJ. * indicates p < 0.05.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: In Vitro, Tube Formation Assay, Microscopy, Quantitation Assay

    Validation of the specificity of Cavin-2 knockdown in HUVECs. HUVEC cells were knocked down with individual siRNAs (numbers 5, 6, 7, and 8) against Cavin-2 ( siCavin-2 ) and non-targeting control ( siControl ). A , WCLs were analyzed in a Western blot for Cavin-2 and β-Actin. Cavin-2 protein expression was severely lost in siRNA numbers 6 and 7. β-Actin serves as a loading control. B , in vitro angiogenesis assay on control and two individual Cavin-2 siRNAs (numbers 6 and 7) in HUVECs show a defective tube formation upon loss of cavin-2. C , graphs represent the quantitation of mean ± S.D. of the number of branches and the total network length in HUVECs computed using ImageJ from three independent experiments. *, indicates p < 0.05.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Validation of the specificity of Cavin-2 knockdown in HUVECs. HUVEC cells were knocked down with individual siRNAs (numbers 5, 6, 7, and 8) against Cavin-2 ( siCavin-2 ) and non-targeting control ( siControl ). A , WCLs were analyzed in a Western blot for Cavin-2 and β-Actin. Cavin-2 protein expression was severely lost in siRNA numbers 6 and 7. β-Actin serves as a loading control. B , in vitro angiogenesis assay on control and two individual Cavin-2 siRNAs (numbers 6 and 7) in HUVECs show a defective tube formation upon loss of cavin-2. C , graphs represent the quantitation of mean ± S.D. of the number of branches and the total network length in HUVECs computed using ImageJ from three independent experiments. *, indicates p < 0.05.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: Western Blot, Expressing, In Vitro, Angiogenesis Assay, Quantitation Assay

    Cavin-2 controls the activity and stability of eNOS. A , WCL of control (siControl) and Cavin-2 (siCavin-2) knocked down HUVECs were separated in SDS-PAGE and immunoblotted for α-eNOS, α-Cavin-2, and α–β-Actin antibodies. B , siControl and siCavin-2 HUVECs were incubated for 10–12 h with endothelial cell growth medium without supplemental growth factors, stimulated with VEGF for activating eNOS, and harvested at the mentioned time points. WCL from harvested cells were immunoblotted for α-phospho-eNOS ( pS1177 ), α-eNOS, α-phospho-p44/42 MAPK at Thr-202/Tyr-204 (pERK), α-p44/42 MAPK (Erk), α-Cavin-2, and α–β-Actin antibodies. β-Actin serves as a loading control. C and D , densitometry-based quantitation of phospho-eNOS ( pS1177 ) ( C ) and total eNOS ( D ) normalized to β-Actin staining for the mentioned time points were performed from two independent experiments using ImageJ. E , live imaging of DAF-2, a cell permeable fluorescence NO indicator, upon VEGF activation on DMSO-treated, l -NAME-treated, siControl, and siCavin-2 HUVECs. The fluorescence emitted were recorded using a confocal microscope and shows a strikingly reduced DAF2 fluorescence in l -NAME-treated and siCavin-2 HUVECs. F , fluorescence signal intensity upon DAF-2 treatment on DMSO-treated, l -NAME-treated, siControl, and siCavin-2 HUVECs were quantified using ImageJ from at least three independent experiments. G , HUVECs were transfected with GFP or Cavin2-GFP plasmids, incubated for 12 h with endothelial cell growth medium without supplemental growth factors, stimulated with or without VEGF, harvested, and immunoblotted for α-phospho-eNOS ( pS1177 ), α-total eNOS, α-GFP, α-Caveolin-1, and α–β-Actin antibodies. Scale bar , 10 μm. * indicates p < 0.05.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Cavin-2 controls the activity and stability of eNOS. A , WCL of control (siControl) and Cavin-2 (siCavin-2) knocked down HUVECs were separated in SDS-PAGE and immunoblotted for α-eNOS, α-Cavin-2, and α–β-Actin antibodies. B , siControl and siCavin-2 HUVECs were incubated for 10–12 h with endothelial cell growth medium without supplemental growth factors, stimulated with VEGF for activating eNOS, and harvested at the mentioned time points. WCL from harvested cells were immunoblotted for α-phospho-eNOS ( pS1177 ), α-eNOS, α-phospho-p44/42 MAPK at Thr-202/Tyr-204 (pERK), α-p44/42 MAPK (Erk), α-Cavin-2, and α–β-Actin antibodies. β-Actin serves as a loading control. C and D , densitometry-based quantitation of phospho-eNOS ( pS1177 ) ( C ) and total eNOS ( D ) normalized to β-Actin staining for the mentioned time points were performed from two independent experiments using ImageJ. E , live imaging of DAF-2, a cell permeable fluorescence NO indicator, upon VEGF activation on DMSO-treated, l -NAME-treated, siControl, and siCavin-2 HUVECs. The fluorescence emitted were recorded using a confocal microscope and shows a strikingly reduced DAF2 fluorescence in l -NAME-treated and siCavin-2 HUVECs. F , fluorescence signal intensity upon DAF-2 treatment on DMSO-treated, l -NAME-treated, siControl, and siCavin-2 HUVECs were quantified using ImageJ from at least three independent experiments. G , HUVECs were transfected with GFP or Cavin2-GFP plasmids, incubated for 12 h with endothelial cell growth medium without supplemental growth factors, stimulated with or without VEGF, harvested, and immunoblotted for α-phospho-eNOS ( pS1177 ), α-total eNOS, α-GFP, α-Caveolin-1, and α–β-Actin antibodies. Scale bar , 10 μm. * indicates p < 0.05.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: Activity Assay, SDS Page, Incubation, Quantitation Assay, Staining, Imaging, Fluorescence, Activation Assay, Microscopy, Transfection

    Caveolae are not required for the regulation of eNOS by Cavin-2. A and B , HUVEC ( A ) and HRMVEC ( B ) were knocked down with control ( siControl ), Cavin-2 ( siCavin-2 ), and Caveolin-1 ( siCaveolin-1 ) using specific siRNAs. Cells were incubated in endothelial cell growth medium without supplemental growth factors for 12 h, stimulated with VEGF, harvested after 15 min, and immunoblotted for α-phospho-eNOS ( pS1177 ), α-total eNOS, α-Cavin2, α-Caveolin-1, and α–β-Actin antibodies. C and D , immunofluorescence analyses of Cavin-2 and Caveolin-1 on HUVECs ( C ) and HRMVECs ( D ) after siControl, siCavin-2, and siCaveolin-1. Continuous staining of caveolin-1 at the plasma membrane is a characteristic of caveolae formation and the DAPI stain represents nuclei. E , ectopic expression and immunoprecipitation ( IP ) of Cavin-2 in HPMEC-ST1.6R cells. Cavin-2-GFP or GFP plasmid were expressed in HPMEC-ST1.6R and the WCL were used for IP. Both, WCL and IP were immunoblotted for antibodies against eNOS, Caveolin-1, and GFP. Scale bar , 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Caveolae are not required for the regulation of eNOS by Cavin-2. A and B , HUVEC ( A ) and HRMVEC ( B ) were knocked down with control ( siControl ), Cavin-2 ( siCavin-2 ), and Caveolin-1 ( siCaveolin-1 ) using specific siRNAs. Cells were incubated in endothelial cell growth medium without supplemental growth factors for 12 h, stimulated with VEGF, harvested after 15 min, and immunoblotted for α-phospho-eNOS ( pS1177 ), α-total eNOS, α-Cavin2, α-Caveolin-1, and α–β-Actin antibodies. C and D , immunofluorescence analyses of Cavin-2 and Caveolin-1 on HUVECs ( C ) and HRMVECs ( D ) after siControl, siCavin-2, and siCaveolin-1. Continuous staining of caveolin-1 at the plasma membrane is a characteristic of caveolae formation and the DAPI stain represents nuclei. E , ectopic expression and immunoprecipitation ( IP ) of Cavin-2 in HPMEC-ST1.6R cells. Cavin-2-GFP or GFP plasmid were expressed in HPMEC-ST1.6R and the WCL were used for IP. Both, WCL and IP were immunoblotted for antibodies against eNOS, Caveolin-1, and GFP. Scale bar , 10 μm.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: Incubation, Immunofluorescence, Staining, Expressing, Immunoprecipitation, Plasmid Preparation

    Cavin-2 controls the cellular localization of eNOS. A , immunofluorescence of α-eNOS antibody on control ( siControl ) and Cavin-2 ( siCavin-2 ) knocked down HUVECs. DAPI stain represents nuclei. B , immunoblots of eNOS and CD31 on total membrane isolated from an equal number of siControl and siCavin-2 HUVECs. Total membranes were resolved along with WCL of untreated HUVECs on SDS-PAGE and transferred to nitrocellulose membrane. On the right side , immunoblots of WCL of siControl and siCavin-2 HUVECs with α-Cavin-2 and α–β-Actin antibodies. β-Actin serves as a loading control. C , scheme of the regulation of NO production by Cavin-2 in endothelial cells. The presence of Cavin-2 positively helps in NO production by stabilizing and activating eNOS. The loss of Cavin-2 adversely effects NO production by destabilizing and inactivating eNOS. Scale bar , 10 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Cavin-2 controls the cellular localization of eNOS. A , immunofluorescence of α-eNOS antibody on control ( siControl ) and Cavin-2 ( siCavin-2 ) knocked down HUVECs. DAPI stain represents nuclei. B , immunoblots of eNOS and CD31 on total membrane isolated from an equal number of siControl and siCavin-2 HUVECs. Total membranes were resolved along with WCL of untreated HUVECs on SDS-PAGE and transferred to nitrocellulose membrane. On the right side , immunoblots of WCL of siControl and siCavin-2 HUVECs with α-Cavin-2 and α–β-Actin antibodies. β-Actin serves as a loading control. C , scheme of the regulation of NO production by Cavin-2 in endothelial cells. The presence of Cavin-2 positively helps in NO production by stabilizing and activating eNOS. The loss of Cavin-2 adversely effects NO production by destabilizing and inactivating eNOS. Scale bar , 10 μm.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: Immunofluorescence, Staining, Western Blot, Isolation, SDS Page

    Cavin-2 is secreted in EMP. A , conditioned media from HUVECs were collected, exosomes and EMP were isolated, separated along with WCL in SDS-PAGE, and immunoblotted to test for the presence of Cavin-2 in exosomes ( TSG101 ), microparticles ( CD31 ), and cytosol (β- Tubulin ). Isolated EMPs were sorted for 0.1 to 1-μm sized particles using nano blank polystyrene beads as reference in flow cytometry. EMPs were analyzed for co-staining of Cavin-2 with endothelial cell surface markers, B and C , VE-cadherin ( B ) and CD31 ( C ). α-Cavin-2 antibody is conjugated with AMCA, both α-VE-cadherin and α-CD31 antibodies were conjugated using PE. D , localization of Cavin-2 on EMPs with α-Cavin-2 antibody followed by goat α-rabbit secondary antibody coupled with 10-nm gold and analyzed using transmission electron microscope. Two representative images from independent experiments were shown. Scale bar , 100 nm. E , EMPs were isolated and concentrated from an equal number of control (siControl) and Cavin-2 (siCavin-2) knockdown HUVECs. Isolated EMPs were separated along with WCL of siControl and siCavin-2 cells in SDS-PAGE, transferred to nitrocellulose membrane, and stained using 0.1% Ponceau S staining reagent. The total protein abundance stained using Ponceau S of EMPs secreted from siControl and siCavin-2 HUVEC were quantified using ImageJ, the signal intensities were plotted from three independent experiments ( a.u., arbitrary units). F , immunoblotting of EMPs and WCLs from siControl and siCavin-2 HUVECs with α-Cavin-2, α-CD31, and α–β-Tubulin antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

    doi: 10.1074/jbc.M117.794743

    Figure Lengend Snippet: Cavin-2 is secreted in EMP. A , conditioned media from HUVECs were collected, exosomes and EMP were isolated, separated along with WCL in SDS-PAGE, and immunoblotted to test for the presence of Cavin-2 in exosomes ( TSG101 ), microparticles ( CD31 ), and cytosol (β- Tubulin ). Isolated EMPs were sorted for 0.1 to 1-μm sized particles using nano blank polystyrene beads as reference in flow cytometry. EMPs were analyzed for co-staining of Cavin-2 with endothelial cell surface markers, B and C , VE-cadherin ( B ) and CD31 ( C ). α-Cavin-2 antibody is conjugated with AMCA, both α-VE-cadherin and α-CD31 antibodies were conjugated using PE. D , localization of Cavin-2 on EMPs with α-Cavin-2 antibody followed by goat α-rabbit secondary antibody coupled with 10-nm gold and analyzed using transmission electron microscope. Two representative images from independent experiments were shown. Scale bar , 100 nm. E , EMPs were isolated and concentrated from an equal number of control (siControl) and Cavin-2 (siCavin-2) knockdown HUVECs. Isolated EMPs were separated along with WCL of siControl and siCavin-2 cells in SDS-PAGE, transferred to nitrocellulose membrane, and stained using 0.1% Ponceau S staining reagent. The total protein abundance stained using Ponceau S of EMPs secreted from siControl and siCavin-2 HUVEC were quantified using ImageJ, the signal intensities were plotted from three independent experiments ( a.u., arbitrary units). F , immunoblotting of EMPs and WCLs from siControl and siCavin-2 HUVECs with α-Cavin-2, α-CD31, and α–β-Tubulin antibodies.

    Article Snippet: The following antibodies were used in this article: α-Cavin-2 (Proteintech), α-phospho-eNOS (Ser-1177), α-phospho-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology), α-eNOS mouse monoclonal (M221) (Abcam), α-β-Actin antibody (Santa Cruz Biotechnology), α-CD31 (WB: Abcam; immunofluorescence: BD Biosciences), α-Caveolin-1 (Abcam), α-GFP (Abcam), α-TSG101 antibody (4A10) (Novus Biologicals), and α,β-Tubulin (Santa Cruz Biotechnology).

    Techniques: Isolation, SDS Page, Flow Cytometry, Staining, Transmission Assay, Microscopy, Western Blot