rabbit anti-vinculin Search Results


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  • 99
    Thermo Fisher vinculin antibody
    Pipox is an endogenous sdRNA-93 target. a Alignment of the putative target site in the Pipox 3′UTR with sdRNA-93. b Pipox LR is specifically repressed by sdRNA-93 in HEK293 transient transfections. Luciferase assays ( n = 4) of HEK293 lysates after cotransfection of Pipox LR, Ctl LR, Anti-93, Anti-Ctl, Mimic-93 and / or Mimic-Ctl as indicated (+ or −). Transfections were normalized to respective LR alone. Pipox LR, luciferase reporter containing sdRNA-93 target site from Pipox 3′UTR; Ctl LR, luciferase reporter containing scrambled sdRNA-93 3′UTR target sites; Anti-93, sdRNA-93 inhibitor; Anti-Ctl, control inhibitor; Mimic-93, sdRNA-93 mimic; Mimic-Ctl, control mimic; RLU, relative light units. c Representative western blots of MDA-MB-231 ( left ) and MCF-7 ( right ) breast cancer cells transfected with sdRNA-93 inhibitor, sdRNA mimic or scrambled control. Pipox and <t>Vinculin</t> (control) blots are shown. Graphs indicate the relative ratio of Pipox to Vinculin as normalized to nontransfected control. ( n ≥ 3)
    Vinculin Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti vinculin
    Localization of MARCKS, α3-integrin, paxillin, and <t>vinculin</t> in three different tumor cell lines on laminin. All images are digitally deconvolved fluorescence micrographs of the attached plasma membrane. For A-C, the first image in each row
    Anti Vinculin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2941 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abcam rabbit anti vinculin
    Focal adhesion proteins are recruited to sites of integrin engagement. (A) Primary human T cells were stimulated on coverslips patterned with ICAM-1 surrounded with OKT3 for 15 min, and labeled with m24 to detect the active, extended-open conformation of LFA-1, and Kim127 to detect the extended form of LFA-1. (B,C) Jurkat T cells were stimulated on VCAM-1 patterns surrounded with anti-CD3 for 15 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin, <t>vinculin,</t> and paxillin. (D,E) Primary human T cells were allowed to interact with VCAM-1 (D) or ICAM-1 (E) patterns surrounded with OKT3 for 17 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin and vinculin. Far right panels in (B–E) show cropped regions in which the four channels have been merged. (E) Scale bars = 10 µm.
    Rabbit Anti Vinculin, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc rabbit anti vinculin
    Arginosuccinate synthase 1 (ASS1) protein is decreased in polycystic kidney disease (PKD). Protein was prepared and immunoblotted for ASS1 and/or β-actin or <t>vinculin.</t> ImageJ or FujiFilm LAS-4000 quantification of protein expression, corrected for the reference proteins, is next to the blots. RNA was extracted, reverse transcribed, and subjected to quantitative PCR for murine Ass1 mRNA (corrected for Eef2, Rpl13a , and Rn18S mRNA levels) or hASS1 mRNA (corrected for PPIA , RPS13 , and RNA18S5 ). Statistically significant differences in ASS1 protein and mRNA expression are indicated on the graphs. Immunoblots are representative of at least two repeats. A : mouse embryonic kidney (MEK) wild-type (WT) and Pkd1 -null (MEK null) cell lines and postnatal mouse Pkd1 -heterozygous (PH2) and Pkd1 -null (PN24) kidney cell lines were harvested for protein and RNA at confluence in triplicate wells. Data are means ± SD. B : protein and RNA were extracted from adult Balb/cJ ( n = 3), day 25 Pkd1 +/+ :Pkhd1-Cre ( Pkd1 +/+ ; n = 3), and day 25 Pkd1 flox/flox :Pkhd1- Cre ( Pkd1 flox/flox ; n = 3) mouse kidneys. Data are means ± SD. C : protein and RNA were extracted from normal human kidneys (NHK; n = 3) and nephrectomy tissue from patients with autosomal dominant PKD (ADPKD) ( n = 6) kidneys. Data are means ± SD.
    Rabbit Anti Vinculin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti vinculin antibody epr8185 loading control
    Arginosuccinate synthase 1 (ASS1) protein is decreased in polycystic kidney disease (PKD). Protein was prepared and immunoblotted for ASS1 and/or β-actin or <t>vinculin.</t> ImageJ or FujiFilm LAS-4000 quantification of protein expression, corrected for the reference proteins, is next to the blots. RNA was extracted, reverse transcribed, and subjected to quantitative PCR for murine Ass1 mRNA (corrected for Eef2, Rpl13a , and Rn18S mRNA levels) or hASS1 mRNA (corrected for PPIA , RPS13 , and RNA18S5 ). Statistically significant differences in ASS1 protein and mRNA expression are indicated on the graphs. Immunoblots are representative of at least two repeats. A : mouse embryonic kidney (MEK) wild-type (WT) and Pkd1 -null (MEK null) cell lines and postnatal mouse Pkd1 -heterozygous (PH2) and Pkd1 -null (PN24) kidney cell lines were harvested for protein and RNA at confluence in triplicate wells. Data are means ± SD. B : protein and RNA were extracted from adult Balb/cJ ( n = 3), day 25 Pkd1 +/+ :Pkhd1-Cre ( Pkd1 +/+ ; n = 3), and day 25 Pkd1 flox/flox :Pkhd1- Cre ( Pkd1 flox/flox ; n = 3) mouse kidneys. Data are means ± SD. C : protein and RNA were extracted from normal human kidneys (NHK; n = 3) and nephrectomy tissue from patients with autosomal dominant PKD (ADPKD) ( n = 6) kidneys. Data are means ± SD.
    Anti Vinculin Antibody Epr8185 Loading Control, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti vinculin
    <t>Vinculin</t> is required for stable F-actin localization within uropods and neutrophil polarization. ( a ) Representative immunoflluorescence images of neutrophils after 30-minute incubation on immobilized ICAM-1 and CXCL1. Cells were stained with Hoescht, phalloidin, and anti-CD11a (n = 3 independent experiments). Scale bar = 10 µm. ( b ) Polarization of neutrophils based on asymmetric F-actin distribution (n = 3 independent experiments). ( c , d ) Background-subtracted and normalized TIRFM fluorescent intensities of F-actin and CD11a (n > 30 cells/group, 3 independent experiments). Analyzed using Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test. ****p
    Anti Vinculin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc vinculin
    <t>Vinculin</t> is required for stable F-actin localization within uropods and neutrophil polarization. ( a ) Representative immunoflluorescence images of neutrophils after 30-minute incubation on immobilized ICAM-1 and CXCL1. Cells were stained with Hoescht, phalloidin, and anti-CD11a (n = 3 independent experiments). Scale bar = 10 µm. ( b ) Polarization of neutrophils based on asymmetric F-actin distribution (n = 3 independent experiments). ( c , d ) Background-subtracted and normalized TIRFM fluorescent intensities of F-actin and CD11a (n > 30 cells/group, 3 independent experiments). Analyzed using Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test. ****p
    Vinculin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 896 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vinculin/product/Cell Signaling Technology Inc
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    89
    Thermo Fisher rabbit anti vinculin
    Redistribution of <t>vinculin</t> (VIN) in HaCaTs in response to chemical and mechanical stimulation. Mechanical stretch applied to cells transduced via focal adhesion in the sites of integrin which initiates downstream signaling. ( a ) Representative images for vinculin in response to DNCB and mechanical stretch. Scale bars are 20 μm. ( b ) VIN length as a function of stretch magnitude. P values were calculated using t-test. * and ** mean P
    Rabbit Anti Vinculin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore monoclonal antivinculin
    Redistribution of <t>vinculin</t> (VIN) in HaCaTs in response to chemical and mechanical stimulation. Mechanical stretch applied to cells transduced via focal adhesion in the sites of integrin which initiates downstream signaling. ( a ) Representative images for vinculin in response to DNCB and mechanical stretch. Scale bars are 20 μm. ( b ) VIN length as a function of stretch magnitude. P values were calculated using t-test. * and ** mean P
    Monoclonal Antivinculin, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti vinculin e1e9v xp rabbit mab
    Redistribution of <t>vinculin</t> (VIN) in HaCaTs in response to chemical and mechanical stimulation. Mechanical stretch applied to cells transduced via focal adhesion in the sites of integrin which initiates downstream signaling. ( a ) Representative images for vinculin in response to DNCB and mechanical stretch. Scale bars are 20 μm. ( b ) VIN length as a function of stretch magnitude. P values were calculated using t-test. * and ** mean P
    Anti Vinculin E1e9v Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rabbit polyclonal anti vinculin
    Redistribution of <t>vinculin</t> (VIN) in HaCaTs in response to chemical and mechanical stimulation. Mechanical stretch applied to cells transduced via focal adhesion in the sites of integrin which initiates downstream signaling. ( a ) Representative images for vinculin in response to DNCB and mechanical stretch. Scale bars are 20 μm. ( b ) VIN length as a function of stretch magnitude. P values were calculated using t-test. * and ** mean P
    Rabbit Polyclonal Anti Vinculin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore vinculin
    Treatment with SM83 reduces the expression of Snai2 both in vivo and in vitro. a NOD/SCID mice engrafted subcutaneously with 5 × 10 6 MDA-MB231 were treated with intraperitoneal injection of SM83 (5 mg/kg, 5 times/week) or left untreated (4 mice/group) until the end of the experiment. Schedule of the experiment (upper panel) and tumor volumes (bottom panel) measured twice a week (significant differences in days 24, 27, and 30. * P = 0.0476, 0.0391, and 0.0344, respectively. Unpaired two-tailed t- test). b Six hours after the last injection, mice were killed, nodules collected, and analyzed by western blot to detect the levels of SM83 targets cIAP1, cIAP2, and XIAP. Actin and <t>vinculin</t> are shown as loading controls. c MDA-MB231 cells transfected in vitro with two siRNAs targeting cIAP1 were treated or not with 100 nM SM83 for 1 h. Western blots were performed to evaluate the levels of cIAP1, cIAP2, and XIAP 72 h after transfection. Values show the fold levels of XIAP. d Differentially expressed genes: heat map showing the 50 genes significantly upregulated and the 15 downregulated by SM83 in MDA-MB231 nodules collected as described in Fig. 1a. e Wound-healing experiments performed with MDA-MB231 cells transfected with control (NT1) or Snai2-specific siRNAs ( n = 4, ** P = 0.0033. Unpaired two-tailed t- test). The graph shows the percentage of gap closure after 24 h of migration. The complete experiment with the other siRNAs tested is shown in Fig. S 2a . f The levels of Snai2, downregulated in the GEP shown in Fig. 1d, and LRIG1, upregulated, were evaluated by western blot performed with lysates of MDA-MB231 nodules. Values show the fold levels of Snai2 and LRIG1 normalized to Actin levels. g Levels of Snai2 in MDA-MB231 cells treated with 100 nM SM83 in time-course experiments. Cleavage of p100 NF-kB2 into the p52 form was used to verify the expected activation of the non-canonical NF-kB pathway upon SM83 administration
    Vinculin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 4488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pipox is an endogenous sdRNA-93 target. a Alignment of the putative target site in the Pipox 3′UTR with sdRNA-93. b Pipox LR is specifically repressed by sdRNA-93 in HEK293 transient transfections. Luciferase assays ( n = 4) of HEK293 lysates after cotransfection of Pipox LR, Ctl LR, Anti-93, Anti-Ctl, Mimic-93 and / or Mimic-Ctl as indicated (+ or −). Transfections were normalized to respective LR alone. Pipox LR, luciferase reporter containing sdRNA-93 target site from Pipox 3′UTR; Ctl LR, luciferase reporter containing scrambled sdRNA-93 3′UTR target sites; Anti-93, sdRNA-93 inhibitor; Anti-Ctl, control inhibitor; Mimic-93, sdRNA-93 mimic; Mimic-Ctl, control mimic; RLU, relative light units. c Representative western blots of MDA-MB-231 ( left ) and MCF-7 ( right ) breast cancer cells transfected with sdRNA-93 inhibitor, sdRNA mimic or scrambled control. Pipox and Vinculin (control) blots are shown. Graphs indicate the relative ratio of Pipox to Vinculin as normalized to nontransfected control. ( n ≥ 3)

    Journal: NPJ Breast Cancer

    Article Title: Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion

    doi: 10.1038/s41523-017-0032-8

    Figure Lengend Snippet: Pipox is an endogenous sdRNA-93 target. a Alignment of the putative target site in the Pipox 3′UTR with sdRNA-93. b Pipox LR is specifically repressed by sdRNA-93 in HEK293 transient transfections. Luciferase assays ( n = 4) of HEK293 lysates after cotransfection of Pipox LR, Ctl LR, Anti-93, Anti-Ctl, Mimic-93 and / or Mimic-Ctl as indicated (+ or −). Transfections were normalized to respective LR alone. Pipox LR, luciferase reporter containing sdRNA-93 target site from Pipox 3′UTR; Ctl LR, luciferase reporter containing scrambled sdRNA-93 3′UTR target sites; Anti-93, sdRNA-93 inhibitor; Anti-Ctl, control inhibitor; Mimic-93, sdRNA-93 mimic; Mimic-Ctl, control mimic; RLU, relative light units. c Representative western blots of MDA-MB-231 ( left ) and MCF-7 ( right ) breast cancer cells transfected with sdRNA-93 inhibitor, sdRNA mimic or scrambled control. Pipox and Vinculin (control) blots are shown. Graphs indicate the relative ratio of Pipox to Vinculin as normalized to nontransfected control. ( n ≥ 3)

    Article Snippet: Alternatively, when possible, membranes were instead simultaneously incubated with antibodies against PIPOX (1:500 dilution) and Vinculin (700062, Invitrogen) (1:5000 dilution) at 4 °C overnight.

    Techniques: Transfection, Luciferase, Cotransfection, CTL Assay, Western Blot, Multiple Displacement Amplification

    Localization of MARCKS, α3-integrin, paxillin, and vinculin in three different tumor cell lines on laminin. All images are digitally deconvolved fluorescence micrographs of the attached plasma membrane. For A-C, the first image in each row

    Journal: Journal of Cell Science

    Article Title: Dynamic adhesions and MARCKS in melanoma cells

    doi: 10.1242/jcs.047860

    Figure Lengend Snippet: Localization of MARCKS, α3-integrin, paxillin, and vinculin in three different tumor cell lines on laminin. All images are digitally deconvolved fluorescence micrographs of the attached plasma membrane. For A-C, the first image in each row

    Article Snippet: Antibodies: anti-MARCKS for western blot from Upstate-Millipore (Billerica, MA) and Santa Cruz Biotechnology (Santa Cruz, CA); anti-MARCKS for immunofluorescence from Proteintech Group (Chicaco, IL); anti-phosphoMARCKS from Cell Signalling Technology (Danvers, MA); anti-α3-integrin from Developmental Studies Hybridoma Bank (University of Iowa, IA); anti-GFP from Abcam (Cambridge, MA); anti-actin and anti-vinculin from Sigma; anti-paxillin from Biosource (Invitrogen, Carlsbad, CA); PKC isoform-specific antibodies from BD Biosciences (Franklin Lakes, NJ); anti-lactate dehydrogenase (LDH) from Rockland (Gilbertsville, PA); horseradish peroxidase (HRP)-conjugated antibody from Vector (Burlingame, CA); all other secondary antibodies from Molecular Probes (Eugene, OR).

    Techniques: Fluorescence

    siRNA-mediated knockdown of Cacna1c prevented lipid peroxidation, but not glutathione depletion following glutamate exposure. a Cacna1c mRNA levels were analyzed 24 h after siRNA transfection with 20 and 40 nM. Gapdh served as internal control. b Protein samples were collected 48 h after transfection with 40 nM siRNA and the Ca V 1.2 expression levels were then identified by Western blot. Vinculin was used as loading control. c Total glutathione levels were calculated from three replicates per condition after 0, 2, 4, and 6 h of glutamate treatment (10 mM). Data are provided as mean + SD. d , e Lipid peroxidation in HT22 cells was determined using BODIPY staining after an 8-h incubation with 9 mM glutamate. The dot plots show representative replicates and the bar graph summarizes the associated experiment where three replicates per sample are shown as percentage of cells in the upper right quarter (mean + SD; 10,000 cells per replicate). Ctrl control, siScr scrambled siRNA, siCacna1c Cacna1c siRNA, Glut glutamate. *** p

    Journal: Cell Death Discovery

    Article Title: Downregulation of the psychiatric susceptibility gene Cacna1c promotes mitochondrial resilience to oxidative stress in neuronal cells

    doi: 10.1038/s41420-018-0061-6

    Figure Lengend Snippet: siRNA-mediated knockdown of Cacna1c prevented lipid peroxidation, but not glutathione depletion following glutamate exposure. a Cacna1c mRNA levels were analyzed 24 h after siRNA transfection with 20 and 40 nM. Gapdh served as internal control. b Protein samples were collected 48 h after transfection with 40 nM siRNA and the Ca V 1.2 expression levels were then identified by Western blot. Vinculin was used as loading control. c Total glutathione levels were calculated from three replicates per condition after 0, 2, 4, and 6 h of glutamate treatment (10 mM). Data are provided as mean + SD. d , e Lipid peroxidation in HT22 cells was determined using BODIPY staining after an 8-h incubation with 9 mM glutamate. The dot plots show representative replicates and the bar graph summarizes the associated experiment where three replicates per sample are shown as percentage of cells in the upper right quarter (mean + SD; 10,000 cells per replicate). Ctrl control, siScr scrambled siRNA, siCacna1c Cacna1c siRNA, Glut glutamate. *** p

    Article Snippet: Vinculin was detected as a loading control using an anti-Vinculin antibody (1:20,000; Sigma-Aldrich, Munich, Germany).

    Techniques: Transfection, Expressing, Western Blot, Staining, Incubation

    Development of NMHC-IIA knockdown-replace cell lines. Representative immunoblots showing expression of endogenous NMHC-IIA, murine GFP-tagged NMHC-IIA and vinculin in parental MDA-MB-231 cells and stable NMHC-IIA knockdown cells.

    Journal: Experimental cell research

    Article Title: Myosin-IIA heavy chain phosphorylation on SI943 regulates tumor metastasis

    doi: 10.1016/j.yexcr.2018.06.028

    Figure Lengend Snippet: Development of NMHC-IIA knockdown-replace cell lines. Representative immunoblots showing expression of endogenous NMHC-IIA, murine GFP-tagged NMHC-IIA and vinculin in parental MDA-MB-231 cells and stable NMHC-IIA knockdown cells.

    Article Snippet: The β-actin and vinculin antibodies were purchased from Sigma.

    Techniques: Western Blot, Expressing, Multiple Displacement Amplification

    Inducible expression of RhoE in RhoE-3T3 cells promotes loss of stress fibers and focal adhesions. RhoE-3T3 cells were grown in the presence (+Tet) or absence (−Tet) of tetracycline. (A) HA-RhoE expression following Tet removal was analyzed by Western blotting with an anti-HA antibody. (B) Fixed RhoE-3T3 cells were stained for F-actin and HA-RhoE with TRITC-labeled phalloidin and an anti-HA antibody, respectively. Bar, 5 μm. (C) Same as panel B, but the cells were stained for F-actin and vinculin with TRITC-labeled phalloidin and an antivinculin antibody, respectively. (D) RhoE-3T3 cells were fixed at the indicated time points after tetracycline removal and stained for F-actin and HA-RhoE with TRITC-labeled phalloidin and an anti-HA antibody, respectively. Bar, 5 μm. Similar results were obtained for three independent clones of RhoE-3T3 cells.

    Journal: Molecular and Cellular Biology

    Article Title: RhoE Inhibits Cell Cycle Progression and Ras-Induced Transformation

    doi: 10.1128/MCB.24.18.7829-7840.2004

    Figure Lengend Snippet: Inducible expression of RhoE in RhoE-3T3 cells promotes loss of stress fibers and focal adhesions. RhoE-3T3 cells were grown in the presence (+Tet) or absence (−Tet) of tetracycline. (A) HA-RhoE expression following Tet removal was analyzed by Western blotting with an anti-HA antibody. (B) Fixed RhoE-3T3 cells were stained for F-actin and HA-RhoE with TRITC-labeled phalloidin and an anti-HA antibody, respectively. Bar, 5 μm. (C) Same as panel B, but the cells were stained for F-actin and vinculin with TRITC-labeled phalloidin and an antivinculin antibody, respectively. (D) RhoE-3T3 cells were fixed at the indicated time points after tetracycline removal and stained for F-actin and HA-RhoE with TRITC-labeled phalloidin and an anti-HA antibody, respectively. Bar, 5 μm. Similar results were obtained for three independent clones of RhoE-3T3 cells.

    Article Snippet: For vinculin staining, cells were incubated for 1 h at 37°C with an antivinculin antibody (v-4505; Sigma) (1:200) followed by 1 h at 37°C with fluorescein-conjugated anti-mouse IgG (Jackson Immunoresearch) (1:200).

    Techniques: Expressing, Western Blot, Staining, Labeling, Clone Assay

    Characterization of total Cx43 in mouse hearts. Western blots of whole heart lysates, isolated from two WT (Wild type-Cx43), CK1 (S325A/S328Y/S330A) or two PKC (S368A) hearts, simultaneously probed for total Cx43. Membranes were also probed with anti-vinculin antibody, as an internal loading control. Notice that Cx43 protein levels are decreased in CK1 and PKC mice compared with WT. Positions of the molecular weight markers are shown on the left

    Journal: Journal of Cardiovascular Disease Research

    Article Title: Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models

    doi: 10.4103/0975-3583.89804

    Figure Lengend Snippet: Characterization of total Cx43 in mouse hearts. Western blots of whole heart lysates, isolated from two WT (Wild type-Cx43), CK1 (S325A/S328Y/S330A) or two PKC (S368A) hearts, simultaneously probed for total Cx43. Membranes were also probed with anti-vinculin antibody, as an internal loading control. Notice that Cx43 protein levels are decreased in CK1 and PKC mice compared with WT. Positions of the molecular weight markers are shown on the left

    Article Snippet: [ ] Cx43 was detected with our Cx43NT1 antibody[ ] and the antibody against vinculin was obtained from Sigma Chemical Co. (V4505).

    Techniques: Western Blot, Isolation, Mouse Assay, Molecular Weight

    Podocyte focal adhesions and actin cytoskeleton. Notes: Representative images of the human podocyte cell line after 2 days of culture. Though cells are not yet arborized, vinculin (left panels) positivity is higher in cells grown on ( A ) collagen-coated plastic and on ( C ) NE surface than in cells adherent to the ( B ) N surface. Phalloidin-rhodamine (middle panels) confirms the preferential organization of the actin cytoskeleton in stress fibers in ( A ) collagen-coated and ( C ) NE surface, whereas peripheral actin prevails in cells grown on ( B ) N material. Merging (right panels), particularly at higher magnification, shows that vinculin co-localizes with the tip of actin filaments in ( A ) and ( C ). Scale bars =10 µm, merge magnification ×3.

    Journal: International Journal of Nanomedicine

    Article Title: A nanoporous surface is essential for glomerular podocyte differentiation in three-dimensional culture

    doi: 10.2147/IJN.S110201

    Figure Lengend Snippet: Podocyte focal adhesions and actin cytoskeleton. Notes: Representative images of the human podocyte cell line after 2 days of culture. Though cells are not yet arborized, vinculin (left panels) positivity is higher in cells grown on ( A ) collagen-coated plastic and on ( C ) NE surface than in cells adherent to the ( B ) N surface. Phalloidin-rhodamine (middle panels) confirms the preferential organization of the actin cytoskeleton in stress fibers in ( A ) collagen-coated and ( C ) NE surface, whereas peripheral actin prevails in cells grown on ( B ) N material. Merging (right panels), particularly at higher magnification, shows that vinculin co-localizes with the tip of actin filaments in ( A ) and ( C ). Scale bars =10 µm, merge magnification ×3.

    Article Snippet: The primary antibodies were rabbit anti-MAP2 (Merck Millipore, Billerica, MA, USA), rabbit anti-vinculin (Sigma-Aldrich Co.), and rabbit anti-β-tubulin III (Sigma-Aldrich Co.).

    Techniques:

    Podocyte focal adhesions and actin cytoskeleton. Notes: After 5 days of culture, the human podocyte cell line grown on ( A ) N surface shows cytoplasmic vinculin with a rounded appearance, typical of the inactive molecule. In contrast, a punctated pattern is present in cells seeded on the ( B ) NE material. ( C ) Vinculin puncta of cells grown on NE surface co-localize with actin tips, as better observed at ( D ) higher magnification (×3). Scale bars =10 µm. The second panels of panels ( A ) and ( B ) represent higher magnification (×4).

    Journal: International Journal of Nanomedicine

    Article Title: A nanoporous surface is essential for glomerular podocyte differentiation in three-dimensional culture

    doi: 10.2147/IJN.S110201

    Figure Lengend Snippet: Podocyte focal adhesions and actin cytoskeleton. Notes: After 5 days of culture, the human podocyte cell line grown on ( A ) N surface shows cytoplasmic vinculin with a rounded appearance, typical of the inactive molecule. In contrast, a punctated pattern is present in cells seeded on the ( B ) NE material. ( C ) Vinculin puncta of cells grown on NE surface co-localize with actin tips, as better observed at ( D ) higher magnification (×3). Scale bars =10 µm. The second panels of panels ( A ) and ( B ) represent higher magnification (×4).

    Article Snippet: The primary antibodies were rabbit anti-MAP2 (Merck Millipore, Billerica, MA, USA), rabbit anti-vinculin (Sigma-Aldrich Co.), and rabbit anti-β-tubulin III (Sigma-Aldrich Co.).

    Techniques:

    A. Talin structure . The modular structure of talin is illustrated and representative binding sites for partners of vertebrate Talin1 are indicated [1, 2, 13]. All known talins, including C. intestinalis talin, contain an N-terminal FERM domain and a C-terminal I/LWEQ module [18]. The FERM domain is a conserved element that links various proteins to the plasma membrane [38]. The I/LWEQ module is a conserved F-actin-binding element that also targets Talin1 to focal adhesions in mammalian cells [13]. We show in this report that the I/LWEQ module of C. intestinalis Talin-a also contains a focal adhesion targeting signal. VBS: vinculin binding sites of Talin1. Scale bar: 500 amino acids. B. Unrooted tree showing the phylogenetic relationships of full-length talins from the mammal Homo sapiens (Hs), the bird Gallus gallus (Gg), the pufferfish Tetraodon nigroviridis (Tn), and Ciona intestinalis (Ci). The vertebrate Talin1 and Talin2 form orthologous groups, with C. intestinalis Talin-a/b as the outgroup. The complete sequence alignment upon which this tree is based and an identity/similarity matrix for these talins are shown in Additional File 1 . Human Talin1 and C. intestinalis Talin-b are 56% identical and 69.4% similar over 2541 amino acids. Scale bar: 10% sequence divergence.

    Journal: BMC Cell Biology

    Article Title: Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

    doi: 10.1186/1471-2121-7-40

    Figure Lengend Snippet: A. Talin structure . The modular structure of talin is illustrated and representative binding sites for partners of vertebrate Talin1 are indicated [1, 2, 13]. All known talins, including C. intestinalis talin, contain an N-terminal FERM domain and a C-terminal I/LWEQ module [18]. The FERM domain is a conserved element that links various proteins to the plasma membrane [38]. The I/LWEQ module is a conserved F-actin-binding element that also targets Talin1 to focal adhesions in mammalian cells [13]. We show in this report that the I/LWEQ module of C. intestinalis Talin-a also contains a focal adhesion targeting signal. VBS: vinculin binding sites of Talin1. Scale bar: 500 amino acids. B. Unrooted tree showing the phylogenetic relationships of full-length talins from the mammal Homo sapiens (Hs), the bird Gallus gallus (Gg), the pufferfish Tetraodon nigroviridis (Tn), and Ciona intestinalis (Ci). The vertebrate Talin1 and Talin2 form orthologous groups, with C. intestinalis Talin-a/b as the outgroup. The complete sequence alignment upon which this tree is based and an identity/similarity matrix for these talins are shown in Additional File 1 . Human Talin1 and C. intestinalis Talin-b are 56% identical and 69.4% similar over 2541 amino acids. Scale bar: 10% sequence divergence.

    Article Snippet: Cells were then fixed with 4% paraformadehyde, permeabilized briefly on ice with 1% Triton X-100, and labeled with either anti-vinculin antibody at a dilution of 1:300 (V9131, Sigma) or FITC-phalloidin at 1:400 dilution as previously described in our analysis of the subcellular targeting of mammalian Talin1 [ ].

    Techniques: Binding Assay, Sequencing

    Subcellular targeting of the I/LWEQ modules of Talin-a and Talin-b . HeLa cells were transiently transfected with either the dsRed-Talin-a.2341–2531 or the dsRed-Talin-b.2341–2531 fusion construct (I/LWEQ module) and counterstained with vinculin to independently label focal adhesions or fluorescein-phalloidin to label F-actin, as previously described [13]. The Talin-a I/LWEQ module targeted to focal adhesions, where the fluorescence signals for dsRed-Talin-a.2341–2531 and the focal adhesion component vinculin overlap (Column 1, overlay). The Talin-a I/LWEQ module did not preferentially localize to actin stress fibers (Column 2). In contrast to Talin-a, the Talin-b I/LWEQ module was not targeted to focal adhesions (Column 3). However, dsRed-Talin-b.2341–2531 did colocalize with actin stress fibers, as shown by the colocalization of the fluorescence signals of dsRed-Talin-b.2341–2531 with phalloidin-stained F-actin (Column 4, overlay).

    Journal: BMC Cell Biology

    Article Title: Evidence that talin alternative splice variants from Ciona intestinalis have different roles in cell adhesion

    doi: 10.1186/1471-2121-7-40

    Figure Lengend Snippet: Subcellular targeting of the I/LWEQ modules of Talin-a and Talin-b . HeLa cells were transiently transfected with either the dsRed-Talin-a.2341–2531 or the dsRed-Talin-b.2341–2531 fusion construct (I/LWEQ module) and counterstained with vinculin to independently label focal adhesions or fluorescein-phalloidin to label F-actin, as previously described [13]. The Talin-a I/LWEQ module targeted to focal adhesions, where the fluorescence signals for dsRed-Talin-a.2341–2531 and the focal adhesion component vinculin overlap (Column 1, overlay). The Talin-a I/LWEQ module did not preferentially localize to actin stress fibers (Column 2). In contrast to Talin-a, the Talin-b I/LWEQ module was not targeted to focal adhesions (Column 3). However, dsRed-Talin-b.2341–2531 did colocalize with actin stress fibers, as shown by the colocalization of the fluorescence signals of dsRed-Talin-b.2341–2531 with phalloidin-stained F-actin (Column 4, overlay).

    Article Snippet: Cells were then fixed with 4% paraformadehyde, permeabilized briefly on ice with 1% Triton X-100, and labeled with either anti-vinculin antibody at a dilution of 1:300 (V9131, Sigma) or FITC-phalloidin at 1:400 dilution as previously described in our analysis of the subcellular targeting of mammalian Talin1 [ ].

    Techniques: Transfection, Construct, Fluorescence, Staining

    EVs from patients with breast cancer induce redistribution of p-FAK and focal adhesions assembly. (A and B) MDA-MB-231 cells cultured on coverslips were treated for 1 h with or without 10 µM of PP2, and stimulated for 30 min with Ctrl EVs and BC EVs. Cells were incubated with Abs against p-FAK, vinculin and tetramethylrhodamine-conjugated phalloidin, and were analyzed by confocal microscopy. (C) Analysis of vinculin in Ctrl EVs and BC EVs by western blotting. (D) Whole cell lysates of MDA-MB-231 cell were included as a control of vinculin expression. Data are presented as the mean ± SD of fluorescent intensities of p-FAK and vinculin, and are expressed as fold above Ctrl. **P

    Journal: Molecular Medicine Reports

    Article Title: Circulating extracellular vesicles from patients with breast cancer enhance migration and invasion via a Src-dependent pathway in MDA-MB-231 breast cancer cells

    doi: 10.3892/mmr.2020.11259

    Figure Lengend Snippet: EVs from patients with breast cancer induce redistribution of p-FAK and focal adhesions assembly. (A and B) MDA-MB-231 cells cultured on coverslips were treated for 1 h with or without 10 µM of PP2, and stimulated for 30 min with Ctrl EVs and BC EVs. Cells were incubated with Abs against p-FAK, vinculin and tetramethylrhodamine-conjugated phalloidin, and were analyzed by confocal microscopy. (C) Analysis of vinculin in Ctrl EVs and BC EVs by western blotting. (D) Whole cell lysates of MDA-MB-231 cell were included as a control of vinculin expression. Data are presented as the mean ± SD of fluorescent intensities of p-FAK and vinculin, and are expressed as fold above Ctrl. **P

    Article Snippet: The membranes were incubated with the following primary antibodies at 4°C overnight: Anti-Flotillin-2 (Flot-2) antibody (Ab; Mouse monoclonal; cat. no. 610383; 1:1,000; BD Biosciences), anti-CD9 Ab C-4 (Mouse monoclonal; cat. no. sc-13118; 1:300; Santa Cruz Biotechnology, Inc.), anti-FAK Ab D-1 (Mouse monoclonal; cat. no. sc-271126; 1:1,000; Santa Cruz Biotechnology, Inc.), anti-c-Src Ab 17AT28 (Mouse monoclonal; cat. no. sc-130124; 1:500; Santa Cruz Biotechnology, Inc.), anti-CD81 Ab EPR4244 (Rabbit monoclonal; cat. no. ab109201; 1:1,000; Abcam), anti-phosphorylated-specific Ab to Tyr-397 of FAK (anti-p-FAK Ab; Rabbit polyclonal; cat. no. 44-624G; IF 1:250; WB 1:1,000; Thermo Fisher Scientific, Inc.), anti-phosphorylated-specific Ab to Tyr-418 of Src (anti-p-Src Ab; rabbit polyclonal IgG; cat. no. AF2685; 1:1,000; R & D Systems, Inc.), anti-vinculin Ab (Rabbit polyclonal; cat. no. V4139; IF 1:250; WB 1:500; Sigma-Aldrich; Merck KGaA) and anti-actin Ab (Mouse polyclonal; 1:1,000) was provided by Dr Manuel Hernandez (Cinvestav-IPN).

    Techniques: Multiple Displacement Amplification, Cell Culture, Incubation, Confocal Microscopy, Western Blot, Expressing

    Focal adhesion proteins are recruited to sites of integrin engagement. (A) Primary human T cells were stimulated on coverslips patterned with ICAM-1 surrounded with OKT3 for 15 min, and labeled with m24 to detect the active, extended-open conformation of LFA-1, and Kim127 to detect the extended form of LFA-1. (B,C) Jurkat T cells were stimulated on VCAM-1 patterns surrounded with anti-CD3 for 15 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin, vinculin, and paxillin. (D,E) Primary human T cells were allowed to interact with VCAM-1 (D) or ICAM-1 (E) patterns surrounded with OKT3 for 17 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin and vinculin. Far right panels in (B–E) show cropped regions in which the four channels have been merged. (E) Scale bars = 10 µm.

    Journal: Frontiers in Immunology

    Article Title: Integrins Modulate T Cell Receptor Signaling by Constraining Actin Flow at the Immunological Synapse

    doi: 10.3389/fimmu.2018.00025

    Figure Lengend Snippet: Focal adhesion proteins are recruited to sites of integrin engagement. (A) Primary human T cells were stimulated on coverslips patterned with ICAM-1 surrounded with OKT3 for 15 min, and labeled with m24 to detect the active, extended-open conformation of LFA-1, and Kim127 to detect the extended form of LFA-1. (B,C) Jurkat T cells were stimulated on VCAM-1 patterns surrounded with anti-CD3 for 15 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin, vinculin, and paxillin. (D,E) Primary human T cells were allowed to interact with VCAM-1 (D) or ICAM-1 (E) patterns surrounded with OKT3 for 17 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin and vinculin. Far right panels in (B–E) show cropped regions in which the four channels have been merged. (E) Scale bars = 10 µm.

    Article Snippet: The following antibodies were used for immunofluorescence microscopy: rabbit anti-vinculin (Cat. #73412) was from Abcam, and rabbit anti-paxillin (Cat. # sc-5574) was from Santa Cruz Biotechnology.

    Techniques: Labeling

    Vinculin and talin couple integrins to actin. (A) in Supplementary Material. (B) Cells generated as in (A) were stimulated on coverslips coated with OKT3 and VCAM-1, and actin flow rates in the LP region were quantified. Mean ± SD were calculated from measurements made in 10–40 cells for each condition, *** p

    Journal: Frontiers in Immunology

    Article Title: Integrins Modulate T Cell Receptor Signaling by Constraining Actin Flow at the Immunological Synapse

    doi: 10.3389/fimmu.2018.00025

    Figure Lengend Snippet: Vinculin and talin couple integrins to actin. (A) in Supplementary Material. (B) Cells generated as in (A) were stimulated on coverslips coated with OKT3 and VCAM-1, and actin flow rates in the LP region were quantified. Mean ± SD were calculated from measurements made in 10–40 cells for each condition, *** p

    Article Snippet: The following antibodies were used for immunofluorescence microscopy: rabbit anti-vinculin (Cat. #73412) was from Abcam, and rabbit anti-paxillin (Cat. # sc-5574) was from Santa Cruz Biotechnology.

    Techniques: Generated, Flow Cytometry

    CuI and CytoD inhibited STAT3 phosphorylation in both signaling complexes through disrupting actin filaments. (A and B) A549 cells were treated with DMSO, CuI (0.5 μmol/L) or CytoD (1 μmol/L) for 2 h, fixed, permeabilized, and stained with DAPI (nucleus, blue) and antibodies against vinculin and p-JAK2 (green) (A) or p-STAT3 (green) (B). Scale bars, 10 μm. (C) Following treatment with DMSO, CuI (0.5 μmol/L) or CytoD (1 μmol/L) for 2 h, A549 cells were lysed and immunoprecipitated for vinculin, and the indicated proteins were detected by immunoblotting using indicated antibodies. (D) A549 cells were treated with DMSO, CuI (0.5 μmol/L) or CytoD (1 μmol/L) for 2 h, followed by stimulation with or without IL-6 (10 ng/mL) for 5 min, and were lysed and immunoprecipitated for GP130. The immunoprecipitates were processed for Western blot analysis. input, 2.5% of the total cell lysates compared with 100% of the pellet; nc, negative control; IgG, the immunoprecipitate of IgG; IP, immunoprecipitation.

    Journal: Acta Pharmacologica Sinica

    Article Title: Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments

    doi: 10.1038/aps.2017.99

    Figure Lengend Snippet: CuI and CytoD inhibited STAT3 phosphorylation in both signaling complexes through disrupting actin filaments. (A and B) A549 cells were treated with DMSO, CuI (0.5 μmol/L) or CytoD (1 μmol/L) for 2 h, fixed, permeabilized, and stained with DAPI (nucleus, blue) and antibodies against vinculin and p-JAK2 (green) (A) or p-STAT3 (green) (B). Scale bars, 10 μm. (C) Following treatment with DMSO, CuI (0.5 μmol/L) or CytoD (1 μmol/L) for 2 h, A549 cells were lysed and immunoprecipitated for vinculin, and the indicated proteins were detected by immunoblotting using indicated antibodies. (D) A549 cells were treated with DMSO, CuI (0.5 μmol/L) or CytoD (1 μmol/L) for 2 h, followed by stimulation with or without IL-6 (10 ng/mL) for 5 min, and were lysed and immunoprecipitated for GP130. The immunoprecipitates were processed for Western blot analysis. input, 2.5% of the total cell lysates compared with 100% of the pellet; nc, negative control; IgG, the immunoprecipitate of IgG; IP, immunoprecipitation.

    Article Snippet: The following primary antibodies were used: rabbit anti-JAK2 (# 3230S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY1007/1008 JAK2 (# 3776S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-STAT3 (# 12640, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY705 STAT3 (# 9145S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-STAT1 (# 14994S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY701 STAT1 (# 7649S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-TYK2 (# 14193S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY1054/1055 TYK2 (# 9321S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-JAK1 (#3332S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY1022/1023 JAK1 (# 3331S, Cell Signaling Technology, Danvers, MA, USA), mouse anti-α-tubulin (# sc5286, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-IL-6Rα (# sc13947, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-GP130 (# sc655, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-SHP2 (# sc280, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-β-actin antibody (# P30002M, Abmart, CA, USA), rabbit anti-vinculin antibody (# ab73412, Abcam, Cambridge, UK), and mouse anti-TCPTP antibody (# PH03L, Calbiochem, Merck Millipore, Billerica, MA, USA).

    Techniques: Staining, Immunoprecipitation, Western Blot, Negative Control

    JAK2/STAT3 phosphorylation were regulated by two signaling complexes: the focal adhesion complex and the IL-6 receptor complex. (A and B) Confocal micrographs of A549 cell staining with DAPI (for nucleus, blue) and antibodies against vinculin (red) and p-JAK2 (green) (A) or p-STAT3 (green) (B). Arrowheads indicated the colocalization of p-JAK2 or p-STAT3 with vinculin. Scale bars, 10 μm. (C) A549 cells were detached by 1 mmol/L EDTA and reattached to plates for various time periods. (D) A549 cells in adhesion (adh) or suspension (sus) were immunoprecipitated for vinculin, followed by immunoblotting with indicated antibodies. (E) A549 cells in adhesion (adh) or suspension (sus) were stimulated with IL-6 (10 ng/mL) for indicated time periods. (F) Confocal micrographs of A549 cell staining with DAPI (for nucleus, blue) and antibodies against vinculin (red) and GP130 (green). Arrows indicated that GP130 and vinculin were not colocalized. Scale bar, 10 μm. (G and H) A549 cell lysates were immunoprecipitated for vinculin, followed by immunoblotting with indicated antibodies. Note that JAK2 and STAT3 bonded with vinculin (H) while GP130 or IL-6Rα did not (G). input, 2.5% of the total cell lysates compared with 100% of the pellet; nc, negative control; IgG, the immunoprecipitate of IgG; IP, immunoprecipitation.

    Journal: Acta Pharmacologica Sinica

    Article Title: Cucurbitacin I inhibits STAT3, but enhances STAT1 signaling in human cancer cells in vitro through disrupting actin filaments

    doi: 10.1038/aps.2017.99

    Figure Lengend Snippet: JAK2/STAT3 phosphorylation were regulated by two signaling complexes: the focal adhesion complex and the IL-6 receptor complex. (A and B) Confocal micrographs of A549 cell staining with DAPI (for nucleus, blue) and antibodies against vinculin (red) and p-JAK2 (green) (A) or p-STAT3 (green) (B). Arrowheads indicated the colocalization of p-JAK2 or p-STAT3 with vinculin. Scale bars, 10 μm. (C) A549 cells were detached by 1 mmol/L EDTA and reattached to plates for various time periods. (D) A549 cells in adhesion (adh) or suspension (sus) were immunoprecipitated for vinculin, followed by immunoblotting with indicated antibodies. (E) A549 cells in adhesion (adh) or suspension (sus) were stimulated with IL-6 (10 ng/mL) for indicated time periods. (F) Confocal micrographs of A549 cell staining with DAPI (for nucleus, blue) and antibodies against vinculin (red) and GP130 (green). Arrows indicated that GP130 and vinculin were not colocalized. Scale bar, 10 μm. (G and H) A549 cell lysates were immunoprecipitated for vinculin, followed by immunoblotting with indicated antibodies. Note that JAK2 and STAT3 bonded with vinculin (H) while GP130 or IL-6Rα did not (G). input, 2.5% of the total cell lysates compared with 100% of the pellet; nc, negative control; IgG, the immunoprecipitate of IgG; IP, immunoprecipitation.

    Article Snippet: The following primary antibodies were used: rabbit anti-JAK2 (# 3230S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY1007/1008 JAK2 (# 3776S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-STAT3 (# 12640, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY705 STAT3 (# 9145S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-STAT1 (# 14994S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY701 STAT1 (# 7649S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-TYK2 (# 14193S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY1054/1055 TYK2 (# 9321S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-JAK1 (#3332S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pY1022/1023 JAK1 (# 3331S, Cell Signaling Technology, Danvers, MA, USA), mouse anti-α-tubulin (# sc5286, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-IL-6Rα (# sc13947, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-GP130 (# sc655, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-SHP2 (# sc280, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-β-actin antibody (# P30002M, Abmart, CA, USA), rabbit anti-vinculin antibody (# ab73412, Abcam, Cambridge, UK), and mouse anti-TCPTP antibody (# PH03L, Calbiochem, Merck Millipore, Billerica, MA, USA).

    Techniques: Staining, Immunoprecipitation, Negative Control

    iExosomes specifically target Kras G12D expression ( A ) KRAS G12D transcript levels in Panc-1 cells (n=3 independent experiments). ( B–C ) 1/Ct values from RT-PCR analysis under the listed conditions, to determine the loading efficiency of siRNA. Standards (siKras G12D , 1:2 and 1:4 dilution): n=1, experimental groups: n=3 independent experiments. ( D ) KRAS G12D transcript levels in Panc-1 cells, n=3 independent experiments. The experiments with 400 exos per cell is the same data that is also presented in panel A. ( E–G ) KRAS G12D transcript levels in Panc-1 cells under the listed conditions. In all groups, n=3 independent experiments. ( H ) Western blotting (Panc-1 cells) for phosphorylated ERK (p-ERK) and Vinculin. si and sh Kras G12D iExo: One way ANOVA, iLipo: two-tailed t-test, n=2 independent experiments. ( I ) RAS pull-down assay. ( J–K ) Panc-1 cells MTT assay (n=5 partitions of indicated treatments with 3 or 6 wells for each partition of treatment) (J) and separate independent experiment (K). ( L–M) TUNEL assay (n=3 distinct wells of Panc-1 cells) (L) and separate independent experiment (M). ( N ) FC analysis of apoptosis in Panc-1 cells. Three different treatments were used to treat n=3 distinct wells of cells. ( O ) Wild-type KRAS transcript levels in BxPC-3 cells (n=3 independent experiments). ( P ) KRAS G12V transcript levels in Capan-1 cells (n=3 independent experiments) ( Q ) KRAS G12C transcript levels in MIA PaCa-2 cells (n=3 independent experiments). ( R–U ) MTT assay: n=5 partitions of treatment given to 3 wells each, BxPC-3 cells ( R ) and separate independent experiment ( S ), n=3 partitions of treatment given to 10 wells each, Capan1 cells ( T ), n=3 partitions of treatment given to 10 wells each, MIA PaCa-2 cells, ( U ). The data is presented as the mean ± SEM. Unless otherwise stated, one-way ANOVA was used to determine statistical significance. * p

    Journal: Nature

    Article Title: Exosomes Facilitate Therapeutic Targeting of Oncogenic Kras in Pancreatic Cancer

    doi: 10.1038/nature22341

    Figure Lengend Snippet: iExosomes specifically target Kras G12D expression ( A ) KRAS G12D transcript levels in Panc-1 cells (n=3 independent experiments). ( B–C ) 1/Ct values from RT-PCR analysis under the listed conditions, to determine the loading efficiency of siRNA. Standards (siKras G12D , 1:2 and 1:4 dilution): n=1, experimental groups: n=3 independent experiments. ( D ) KRAS G12D transcript levels in Panc-1 cells, n=3 independent experiments. The experiments with 400 exos per cell is the same data that is also presented in panel A. ( E–G ) KRAS G12D transcript levels in Panc-1 cells under the listed conditions. In all groups, n=3 independent experiments. ( H ) Western blotting (Panc-1 cells) for phosphorylated ERK (p-ERK) and Vinculin. si and sh Kras G12D iExo: One way ANOVA, iLipo: two-tailed t-test, n=2 independent experiments. ( I ) RAS pull-down assay. ( J–K ) Panc-1 cells MTT assay (n=5 partitions of indicated treatments with 3 or 6 wells for each partition of treatment) (J) and separate independent experiment (K). ( L–M) TUNEL assay (n=3 distinct wells of Panc-1 cells) (L) and separate independent experiment (M). ( N ) FC analysis of apoptosis in Panc-1 cells. Three different treatments were used to treat n=3 distinct wells of cells. ( O ) Wild-type KRAS transcript levels in BxPC-3 cells (n=3 independent experiments). ( P ) KRAS G12V transcript levels in Capan-1 cells (n=3 independent experiments) ( Q ) KRAS G12C transcript levels in MIA PaCa-2 cells (n=3 independent experiments). ( R–U ) MTT assay: n=5 partitions of treatment given to 3 wells each, BxPC-3 cells ( R ) and separate independent experiment ( S ), n=3 partitions of treatment given to 10 wells each, Capan1 cells ( T ), n=3 partitions of treatment given to 10 wells each, MIA PaCa-2 cells, ( U ). The data is presented as the mean ± SEM. Unless otherwise stated, one-way ANOVA was used to determine statistical significance. * p

    Article Snippet: The membranes were then blocked for 1 hour at room temperature with 5% non-fat dry milk in PBS with 0.05% Tween-20, and incubated overnight at 4°C with the following primary antibodies: anti-rabbit p-Erk-p44/p42 MAPK (Erk1/2) (Thr202/Tyr 204) (Cell Signaling, 4376, 1:1,000), anti-rabbit Vinculin (Abcam, 129002, 1:10,000).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test, Pull Down Assay, MTT Assay, TUNEL Assay

    Arginosuccinate synthase 1 (ASS1) protein is decreased in polycystic kidney disease (PKD). Protein was prepared and immunoblotted for ASS1 and/or β-actin or vinculin. ImageJ or FujiFilm LAS-4000 quantification of protein expression, corrected for the reference proteins, is next to the blots. RNA was extracted, reverse transcribed, and subjected to quantitative PCR for murine Ass1 mRNA (corrected for Eef2, Rpl13a , and Rn18S mRNA levels) or hASS1 mRNA (corrected for PPIA , RPS13 , and RNA18S5 ). Statistically significant differences in ASS1 protein and mRNA expression are indicated on the graphs. Immunoblots are representative of at least two repeats. A : mouse embryonic kidney (MEK) wild-type (WT) and Pkd1 -null (MEK null) cell lines and postnatal mouse Pkd1 -heterozygous (PH2) and Pkd1 -null (PN24) kidney cell lines were harvested for protein and RNA at confluence in triplicate wells. Data are means ± SD. B : protein and RNA were extracted from adult Balb/cJ ( n = 3), day 25 Pkd1 +/+ :Pkhd1-Cre ( Pkd1 +/+ ; n = 3), and day 25 Pkd1 flox/flox :Pkhd1- Cre ( Pkd1 flox/flox ; n = 3) mouse kidneys. Data are means ± SD. C : protein and RNA were extracted from normal human kidneys (NHK; n = 3) and nephrectomy tissue from patients with autosomal dominant PKD (ADPKD) ( n = 6) kidneys. Data are means ± SD.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis

    doi: 10.1152/ajprenal.00025.2018

    Figure Lengend Snippet: Arginosuccinate synthase 1 (ASS1) protein is decreased in polycystic kidney disease (PKD). Protein was prepared and immunoblotted for ASS1 and/or β-actin or vinculin. ImageJ or FujiFilm LAS-4000 quantification of protein expression, corrected for the reference proteins, is next to the blots. RNA was extracted, reverse transcribed, and subjected to quantitative PCR for murine Ass1 mRNA (corrected for Eef2, Rpl13a , and Rn18S mRNA levels) or hASS1 mRNA (corrected for PPIA , RPS13 , and RNA18S5 ). Statistically significant differences in ASS1 protein and mRNA expression are indicated on the graphs. Immunoblots are representative of at least two repeats. A : mouse embryonic kidney (MEK) wild-type (WT) and Pkd1 -null (MEK null) cell lines and postnatal mouse Pkd1 -heterozygous (PH2) and Pkd1 -null (PN24) kidney cell lines were harvested for protein and RNA at confluence in triplicate wells. Data are means ± SD. B : protein and RNA were extracted from adult Balb/cJ ( n = 3), day 25 Pkd1 +/+ :Pkhd1-Cre ( Pkd1 +/+ ; n = 3), and day 25 Pkd1 flox/flox :Pkhd1- Cre ( Pkd1 flox/flox ; n = 3) mouse kidneys. Data are means ± SD. C : protein and RNA were extracted from normal human kidneys (NHK; n = 3) and nephrectomy tissue from patients with autosomal dominant PKD (ADPKD) ( n = 6) kidneys. Data are means ± SD.

    Article Snippet: The antibodies used were monoclonal (2B10) mouse anti-ASS1 antibody (Invitrogen, Carlsbad, CA), rabbit anti-vinculin (Cell Signaling Technology, Danvers, MA), and rabbit anti-β-actin (Cell Signaling Technology).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    Murine arginosuccinate synthase 1 (mASS1) expression is significantly decreased by day 21 in the Pkd1 flox/flox :Pkhd1- Cre mouse model of polycystic kidney disease (PKD). Protein was extracted from postnatal day 10 (P10), P14, and P21 Pkd1 +/+ :Pkhd1-Cre ( Pkd1 +/+ ; n = 3), Pkd1 +/flox :Pkhd1-Cre ( Pkd1 +/flox ; n = 3), and Pkd1 flox/flox :Pkhd1- Cre ( Pkd1 flox/flox ; n = 3) mouse kidneys and immunoblotted for mASS1 and/or β-actin or vinculin. Quantification of ASS1 using the FujiFilm LAS-4000, corrected for the housekeeping protein, is next to each blot.

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis

    doi: 10.1152/ajprenal.00025.2018

    Figure Lengend Snippet: Murine arginosuccinate synthase 1 (mASS1) expression is significantly decreased by day 21 in the Pkd1 flox/flox :Pkhd1- Cre mouse model of polycystic kidney disease (PKD). Protein was extracted from postnatal day 10 (P10), P14, and P21 Pkd1 +/+ :Pkhd1-Cre ( Pkd1 +/+ ; n = 3), Pkd1 +/flox :Pkhd1-Cre ( Pkd1 +/flox ; n = 3), and Pkd1 flox/flox :Pkhd1- Cre ( Pkd1 flox/flox ; n = 3) mouse kidneys and immunoblotted for mASS1 and/or β-actin or vinculin. Quantification of ASS1 using the FujiFilm LAS-4000, corrected for the housekeeping protein, is next to each blot.

    Article Snippet: The antibodies used were monoclonal (2B10) mouse anti-ASS1 antibody (Invitrogen, Carlsbad, CA), rabbit anti-vinculin (Cell Signaling Technology, Danvers, MA), and rabbit anti-β-actin (Cell Signaling Technology).

    Techniques: Expressing

    Vinculin protein expression in A10 cells cultured on matrices with different stiffness: 2, 8, 50 kPa, and polystrene ( > GPa). A) western blot of A10 samples: supernatant fractions (Lanes 1–4), cytoskeleton fractions (Lanes 5–8), recombinant vinculin (Lane 9), and chicken gizzard muscle extract (Lane 10). Vinculin bands (~ 130 kDa) and metavinculin band (~150 kDa) were labeled with arrows. 10 μg of total protein sample was loaded on each lane. B) The amount of vinculin in supernatant fractions relative to GAPDH. Data are presented as mean ± SD. * Denotes p value

    Journal: Biochemical and biophysical research communications

    Article Title: The Role of Extracellular Matrix Stiffness in Regulating Cytoskeletal Remodeling via Vinculin in Synthetic Smooth Muscle Cells

    doi: 10.1016/j.bbrc.2018.11.142

    Figure Lengend Snippet: Vinculin protein expression in A10 cells cultured on matrices with different stiffness: 2, 8, 50 kPa, and polystrene ( > GPa). A) western blot of A10 samples: supernatant fractions (Lanes 1–4), cytoskeleton fractions (Lanes 5–8), recombinant vinculin (Lane 9), and chicken gizzard muscle extract (Lane 10). Vinculin bands (~ 130 kDa) and metavinculin band (~150 kDa) were labeled with arrows. 10 μg of total protein sample was loaded on each lane. B) The amount of vinculin in supernatant fractions relative to GAPDH. Data are presented as mean ± SD. * Denotes p value

    Article Snippet: Cells were then fixed, permeabilized, and stained with rabbit anti-vinculin antibody (Cell Signaling Technology) overnight at 4°C.

    Techniques: Expressing, Cell Culture, Western Blot, Recombinant, Labeling

    Gene expression levels of vinculin (Vcl) relative to that of the reference gene HPRT1 in A10 cells cultured on matrices with different stiffness. Data are presented as mean ± SD. * Denotes P value

    Journal: Biochemical and biophysical research communications

    Article Title: The Role of Extracellular Matrix Stiffness in Regulating Cytoskeletal Remodeling via Vinculin in Synthetic Smooth Muscle Cells

    doi: 10.1016/j.bbrc.2018.11.142

    Figure Lengend Snippet: Gene expression levels of vinculin (Vcl) relative to that of the reference gene HPRT1 in A10 cells cultured on matrices with different stiffness. Data are presented as mean ± SD. * Denotes P value

    Article Snippet: Cells were then fixed, permeabilized, and stained with rabbit anti-vinculin antibody (Cell Signaling Technology) overnight at 4°C.

    Techniques: Expressing, Cell Culture

    Immunofluorescent images of A10 cells cultured on collagen coated matrices with selected stiffness. Actin was stained with phalloidin (red) while vinculin was visualized in green. scale bar: 10 μm.

    Journal: Biochemical and biophysical research communications

    Article Title: The Role of Extracellular Matrix Stiffness in Regulating Cytoskeletal Remodeling via Vinculin in Synthetic Smooth Muscle Cells

    doi: 10.1016/j.bbrc.2018.11.142

    Figure Lengend Snippet: Immunofluorescent images of A10 cells cultured on collagen coated matrices with selected stiffness. Actin was stained with phalloidin (red) while vinculin was visualized in green. scale bar: 10 μm.

    Article Snippet: Cells were then fixed, permeabilized, and stained with rabbit anti-vinculin antibody (Cell Signaling Technology) overnight at 4°C.

    Techniques: Cell Culture, Staining

    Vinculin is required for stable F-actin localization within uropods and neutrophil polarization. ( a ) Representative immunoflluorescence images of neutrophils after 30-minute incubation on immobilized ICAM-1 and CXCL1. Cells were stained with Hoescht, phalloidin, and anti-CD11a (n = 3 independent experiments). Scale bar = 10 µm. ( b ) Polarization of neutrophils based on asymmetric F-actin distribution (n = 3 independent experiments). ( c , d ) Background-subtracted and normalized TIRFM fluorescent intensities of F-actin and CD11a (n > 30 cells/group, 3 independent experiments). Analyzed using Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test. ****p

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Vinculin is required for stable F-actin localization within uropods and neutrophil polarization. ( a ) Representative immunoflluorescence images of neutrophils after 30-minute incubation on immobilized ICAM-1 and CXCL1. Cells were stained with Hoescht, phalloidin, and anti-CD11a (n = 3 independent experiments). Scale bar = 10 µm. ( b ) Polarization of neutrophils based on asymmetric F-actin distribution (n = 3 independent experiments). ( c , d ) Background-subtracted and normalized TIRFM fluorescent intensities of F-actin and CD11a (n > 30 cells/group, 3 independent experiments). Analyzed using Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test. ****p

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques: Incubation, Staining

    Vinculin plays a role in β2 integrin-dependent neutrophil motility. ( a – d ) Parameters of neutrophil motility during 30-minute chemokinesis on immobilized ICAM-1 and CXCL1 (n > 160 cells/group, 3 independent experiments). Analyzed using unpaired Student’s t-test. **p

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Vinculin plays a role in β2 integrin-dependent neutrophil motility. ( a – d ) Parameters of neutrophil motility during 30-minute chemokinesis on immobilized ICAM-1 and CXCL1 (n > 160 cells/group, 3 independent experiments). Analyzed using unpaired Student’s t-test. **p

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques:

    Neutrophil traction stress generation is attenuated by vinculin deficiency. ( a ) 3D tractions and the trace of the dipole moments, µ, of WT and VclKO neutrophils on polyacrylamide gels of either 0.5 kPa or 1.5 kPa stiffness (n > 25 cells/group, 3 independent experiments). Data analyzed using two-way ANOVA with Tukey multiple comparison test. *p

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Neutrophil traction stress generation is attenuated by vinculin deficiency. ( a ) 3D tractions and the trace of the dipole moments, µ, of WT and VclKO neutrophils on polyacrylamide gels of either 0.5 kPa or 1.5 kPa stiffness (n > 25 cells/group, 3 independent experiments). Data analyzed using two-way ANOVA with Tukey multiple comparison test. *p

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques:

    Vinculin is dispensable for neutrophil motility under shear stress. ( a – d ) Parameters of neutrophil motility during 60-minute chemokinesis in a flow chamber coated with E-selectin, ICAM-1 and CXCL1, and perfused at a wall shear stress of 2 dyne/cm 2 (n > 70 cells/group, 5 replicate runs, 2 independent experiments). Analyzed using unpaired Student’s t-test. ( e ) Neutrophil tracks during 60-minute chemokinesis in a flow chamber (as in a – d ).

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Vinculin is dispensable for neutrophil motility under shear stress. ( a – d ) Parameters of neutrophil motility during 60-minute chemokinesis in a flow chamber coated with E-selectin, ICAM-1 and CXCL1, and perfused at a wall shear stress of 2 dyne/cm 2 (n > 70 cells/group, 5 replicate runs, 2 independent experiments). Analyzed using unpaired Student’s t-test. ( e ) Neutrophil tracks during 60-minute chemokinesis in a flow chamber (as in a – d ).

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques:

    Vinculin is dispensable for neutrophil recruitment in vivo . ( a ) Percentage composition of control (Vcl f/f ) and vinculin knockout (Vcl f/f MX1 cre ) neutrophils in the peripheral blood and peritoneal lavage, 4 hours after induction of peritonitis in mixed chimeric mice (n = 5 mice, 2 independent experiments). Analyzed using two-way ANOVA with Tukey multiple comparison test. ( b , c ) The arrest and time course of sustained adhesion of neutrophils in response to intravenous injection of CXCL1, and over the 15-minute period immediately following (n = 12 fields of view, across 7 chimeric mice). Data were analyzed using non-linear regression: (WT) Y = e −0.119t , (VclKO) Y = e −0.136t . ( d ) Soluble ICAM-1 binding to bone marrow neutrophils in response to CXCL1, as measured by flow cytometry (3 replicates per group, n = 3 independent experiments).

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Vinculin is dispensable for neutrophil recruitment in vivo . ( a ) Percentage composition of control (Vcl f/f ) and vinculin knockout (Vcl f/f MX1 cre ) neutrophils in the peripheral blood and peritoneal lavage, 4 hours after induction of peritonitis in mixed chimeric mice (n = 5 mice, 2 independent experiments). Analyzed using two-way ANOVA with Tukey multiple comparison test. ( b , c ) The arrest and time course of sustained adhesion of neutrophils in response to intravenous injection of CXCL1, and over the 15-minute period immediately following (n = 12 fields of view, across 7 chimeric mice). Data were analyzed using non-linear regression: (WT) Y = e −0.119t , (VclKO) Y = e −0.136t . ( d ) Soluble ICAM-1 binding to bone marrow neutrophils in response to CXCL1, as measured by flow cytometry (3 replicates per group, n = 3 independent experiments).

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques: In Vivo, Knock-Out, Mouse Assay, Injection, Binding Assay, Flow Cytometry

    Vinculin knockout attenuates β2 integrin-dependent neutrophil adhesion. ( a ) Adhesion of neutrophils to immobilized ICAM-1 and CXCL1 assessed for progenitor-derived wild-type (WT), vinculin knockout (VclKO) created using sgRNAs (1) and (2), β2 integrin knockout (Itgb2KO), and murine bone marrow neutrophils (n = 3 independent experiments). Analyzed using one-way ANOVA with Tukey pairwise multiple comparison test. **p

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Vinculin knockout attenuates β2 integrin-dependent neutrophil adhesion. ( a ) Adhesion of neutrophils to immobilized ICAM-1 and CXCL1 assessed for progenitor-derived wild-type (WT), vinculin knockout (VclKO) created using sgRNAs (1) and (2), β2 integrin knockout (Itgb2KO), and murine bone marrow neutrophils (n = 3 independent experiments). Analyzed using one-way ANOVA with Tukey pairwise multiple comparison test. **p

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques: Knock-Out, Derivative Assay

    Vinculin plays a role in neutrophil mechanosensing. ( a , b ) Neutrophil cell area and spreading frequency on polyacrylamide gels of varying stiffness (soft: 1.5 kPa, intermediate: 8.3 kPa, stiff: 100 kPa) conjugated with ICAM-1 and CXCL1 (n > 100 cells/group, 15 fields of view/group, 3 independent experiments). Analyzed using two-way ANOVA with Tukey multiple comparison test. **p

    Journal: Scientific Reports

    Article Title: Context-Dependent Role of Vinculin in Neutrophil Adhesion, Motility and Trafficking

    doi: 10.1038/s41598-020-58882-y

    Figure Lengend Snippet: Vinculin plays a role in neutrophil mechanosensing. ( a , b ) Neutrophil cell area and spreading frequency on polyacrylamide gels of varying stiffness (soft: 1.5 kPa, intermediate: 8.3 kPa, stiff: 100 kPa) conjugated with ICAM-1 and CXCL1 (n > 100 cells/group, 15 fields of view/group, 3 independent experiments). Analyzed using two-way ANOVA with Tukey multiple comparison test. **p

    Article Snippet: Antibodies: anti-CD18 (clone GAME-46; BD Biosciences), anti-CD11a (clone M17/4; BioLegend), anti-ICAM-1 (clone YN1; BioLegend), APC-anti-CD11b (clone M1/70; BioLegend), anti-Ly6G (clone 1A8; BioLegend), APC-anti-CD117 (clone 2B8; BioLegend), anti-CXCR2 (clone SA045E1; BioLegend), anti-α-actinin (Cell Signaling Technologies), anti-vinculin (Cell Signaling Technologies), anti-GFP (Cell Signaling Technologies), HRP-conjugated-anti-Rabbit IgG (Cell Signaling Technologies), anti-CD11a (clone IBL-6/2; Cell Signaling Techologies), Alexa Fluor 647-anti-Rat IgG (ThermoFisher Scientific).

    Techniques:

    Redistribution of vinculin (VIN) in HaCaTs in response to chemical and mechanical stimulation. Mechanical stretch applied to cells transduced via focal adhesion in the sites of integrin which initiates downstream signaling. ( a ) Representative images for vinculin in response to DNCB and mechanical stretch. Scale bars are 20 μm. ( b ) VIN length as a function of stretch magnitude. P values were calculated using t-test. * and ** mean P

    Journal: Scientific Reports

    Article Title: Effect of Mechanical Stretch on the DNCB-induced Proinflammatory Cytokine Secretion in Human Keratinocytes

    doi: 10.1038/s41598-019-41480-y

    Figure Lengend Snippet: Redistribution of vinculin (VIN) in HaCaTs in response to chemical and mechanical stimulation. Mechanical stretch applied to cells transduced via focal adhesion in the sites of integrin which initiates downstream signaling. ( a ) Representative images for vinculin in response to DNCB and mechanical stretch. Scale bars are 20 μm. ( b ) VIN length as a function of stretch magnitude. P values were calculated using t-test. * and ** mean P

    Article Snippet: For focal adhesion staining, cells were incubated with rabbit anti-vinculin (Invitrogen) primary antibody for 1 h at room temperature.

    Techniques:

    Treatment with SM83 reduces the expression of Snai2 both in vivo and in vitro. a NOD/SCID mice engrafted subcutaneously with 5 × 10 6 MDA-MB231 were treated with intraperitoneal injection of SM83 (5 mg/kg, 5 times/week) or left untreated (4 mice/group) until the end of the experiment. Schedule of the experiment (upper panel) and tumor volumes (bottom panel) measured twice a week (significant differences in days 24, 27, and 30. * P = 0.0476, 0.0391, and 0.0344, respectively. Unpaired two-tailed t- test). b Six hours after the last injection, mice were killed, nodules collected, and analyzed by western blot to detect the levels of SM83 targets cIAP1, cIAP2, and XIAP. Actin and vinculin are shown as loading controls. c MDA-MB231 cells transfected in vitro with two siRNAs targeting cIAP1 were treated or not with 100 nM SM83 for 1 h. Western blots were performed to evaluate the levels of cIAP1, cIAP2, and XIAP 72 h after transfection. Values show the fold levels of XIAP. d Differentially expressed genes: heat map showing the 50 genes significantly upregulated and the 15 downregulated by SM83 in MDA-MB231 nodules collected as described in Fig. 1a. e Wound-healing experiments performed with MDA-MB231 cells transfected with control (NT1) or Snai2-specific siRNAs ( n = 4, ** P = 0.0033. Unpaired two-tailed t- test). The graph shows the percentage of gap closure after 24 h of migration. The complete experiment with the other siRNAs tested is shown in Fig. S 2a . f The levels of Snai2, downregulated in the GEP shown in Fig. 1d, and LRIG1, upregulated, were evaluated by western blot performed with lysates of MDA-MB231 nodules. Values show the fold levels of Snai2 and LRIG1 normalized to Actin levels. g Levels of Snai2 in MDA-MB231 cells treated with 100 nM SM83 in time-course experiments. Cleavage of p100 NF-kB2 into the p52 form was used to verify the expected activation of the non-canonical NF-kB pathway upon SM83 administration

    Journal: Cell Death and Differentiation

    Article Title: cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells

    doi: 10.1038/s41418-018-0100-0

    Figure Lengend Snippet: Treatment with SM83 reduces the expression of Snai2 both in vivo and in vitro. a NOD/SCID mice engrafted subcutaneously with 5 × 10 6 MDA-MB231 were treated with intraperitoneal injection of SM83 (5 mg/kg, 5 times/week) or left untreated (4 mice/group) until the end of the experiment. Schedule of the experiment (upper panel) and tumor volumes (bottom panel) measured twice a week (significant differences in days 24, 27, and 30. * P = 0.0476, 0.0391, and 0.0344, respectively. Unpaired two-tailed t- test). b Six hours after the last injection, mice were killed, nodules collected, and analyzed by western blot to detect the levels of SM83 targets cIAP1, cIAP2, and XIAP. Actin and vinculin are shown as loading controls. c MDA-MB231 cells transfected in vitro with two siRNAs targeting cIAP1 were treated or not with 100 nM SM83 for 1 h. Western blots were performed to evaluate the levels of cIAP1, cIAP2, and XIAP 72 h after transfection. Values show the fold levels of XIAP. d Differentially expressed genes: heat map showing the 50 genes significantly upregulated and the 15 downregulated by SM83 in MDA-MB231 nodules collected as described in Fig. 1a. e Wound-healing experiments performed with MDA-MB231 cells transfected with control (NT1) or Snai2-specific siRNAs ( n = 4, ** P = 0.0033. Unpaired two-tailed t- test). The graph shows the percentage of gap closure after 24 h of migration. The complete experiment with the other siRNAs tested is shown in Fig. S 2a . f The levels of Snai2, downregulated in the GEP shown in Fig. 1d, and LRIG1, upregulated, were evaluated by western blot performed with lysates of MDA-MB231 nodules. Values show the fold levels of Snai2 and LRIG1 normalized to Actin levels. g Levels of Snai2 in MDA-MB231 cells treated with 100 nM SM83 in time-course experiments. Cleavage of p100 NF-kB2 into the p52 form was used to verify the expected activation of the non-canonical NF-kB pathway upon SM83 administration

    Article Snippet: Membranes were then saturated for 30 min in Tris-buffered saline containing 4% BSA and incubated overnight with the anti-human primary antibodies recognizing cIAP1 #ab108361, total EGFR #ab30 (Abcam); Snai2 #9585, LRIG1 #12752, NF-kB2 #4882, NF-kB1 #3035, Cleaved Caspase3 #9501, p65/RelA #3034, pEGFR Tyr1068 #2236, c-Cbl #2747, Myc-Tag #2278 (Cell Signaling Technology, Danvers, MA, USA); phospho-ERK1/2 #M8159, ERK1/2 #M5670, Actin #A1978 and Vinculin #V9131 (Sigma-Aldrich), cIAP2 #552783 and XIAP #610763 (BD Biosciences).

    Techniques: Expressing, In Vivo, In Vitro, Mouse Assay, Multiple Displacement Amplification, Injection, Two Tailed Test, Western Blot, Transfection, Migration, Activation Assay