700062

antibody from Invitrogen Antibodies

Targeting: VCL

Provider product page for 700062

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [38]
Comments [0]
Validations
Western blot [5]
Immunocytochemistry [3]
Flow cytometry [1]
Other assay [20]

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Product number: 700062 - Provider product page
Provider: Invitrogen Antibodies
Product name: Vinculin Recombinant Rabbit Monoclonal Antibody (42H89L44)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Antibody clone number: 42H89L44
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

VCL protein structure - 700062 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Vinculin in HeLa cell lysate using a Vinculin recombinant rabbit monoclonal antibody (Product # 700062) at a dilution of 0.5 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Vinculin was performed by loading 20 µg of HeLa (lane1) and NIH/3T3 (lane2) cell lysates using Novex®NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Proteins were transferred to a PVDF membrane and blocked with 5 % skim milk for 1 hour at room temperature. Vinculin was detected at ~125 kDa using Vinculin Recombinant Rabbit Monoclonal Antibody (Product # 700062) at 1 µg-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Vinculin in HeLa cell lysate using a Vinculin recombinant rabbit monoclonal antibody (Product # 700062) at a dilution of 0.5 µg/mL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Vinculin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR948199_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Vinculin was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa Vinculin KO (Lane 3) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Vinculin Recombinant Rabbit Monoclonal Antibody (42H89L44) (Product # 700062, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:6,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Vinculin. Anuncharacterized band observed at ~55 kDa in all the samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Vinculin Recombinant Rabbit Monoclonal Antibody (42H89L44)(Product # 700062) and a 124kDa band corresponding to Vinculin was observed across cell lines and tissues tested. Membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), NIH/3T3 (Lane 2), U-87 MG (Lane 3), PC-3 (Lane 4), MCF-7 (Lane 5), MOLT-4 (Lane 6), Jurkat (Lane 7). Tissue extracts of Mouse Lung (Lane 8), Rat Lung (Lane 9), Mouse Brain (Lane 10), Rat Brain (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1ug/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Vinculin in HeLa cells using a Vinculin recombinant rabbit monoclonal antibody (Product # 700062) at a dilution of 1 µg/mL (left) or preincubated with the recombinant protein used as an immunogen (right), followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000. Nuclei are stained with Hoescht (blue) and actin stained a fluorescent red phalloidin. Note punctate staining at cell-cell and cell-matrix junctions.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Vinculin was performed using 70 confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Vinculin Recombinant Rabbit Monoclonal Antibody (42H89L44) (Product # 700062) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cytoskeleton along with focal adhesion signal which is also reported localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Vinculin was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes; permeabilized with 0.25% Triton X-100 for 10 minutes followed by blocking with 5% BSA for 1 hour at room temperature. The cells were incubated with Vinculin Recombinant Rabbit Monoclonal Antibody (Product # 700062) at 2 µg-4 µg in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing junction and cytoplasmic localization of Vinculin. Panel e shows no primary antibody control. The images were captured at 20X magnification.

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 In DCs, the ASAP1 protein is partly associated with podosomes and its depletion leads to impaired matrix degradation and migration ( a ) Confocal microscopy showing ASAP1 (green), actin (red), vinculin (blue) and nuclei (white) in DCs derived from monocytes of a healthy volunteer. Podosomes are structures characterized by the actin core (visible as red dots) surrounded by other proteins. Boxed area is shown in grayscale enlarged on the right. Scale bar represents 7.5mum. ( b ) Representative confocal microscopy images (left panel) showing monocyte-derived DCs treated with either ASAP1 or control siRNA and seeded on FITC-conjugated gelatine (green) for 4 hours. The cells were stained to visualize actin (phalloidin-red) and nuclei (DAPI-blue). White arrows show examples of the DAPI-stained nuclei of cells that do not degrade matrix. Scale bar represents 50mum. In the same experiment cell lysates were collected for western blotting and probed with anti-ASAP1 and anti-actin antibodies (right panel) to assess knockdown efficiency. Area of degradation of the FITC-conjugated gelatine matrix by DCs (lower panel). In total 394 and 317 DCs treated with either ASAP1 or control siRNA, respectively, were counted in 4 independent experiments. ( c ) Migration velocity of DCs in a Dunn chamber. In total 162 and 186 DCs treated with either ASAP1 or control siRNA, respectively, were tracked in 5 independent experiments. Graphs show mean values +- SEM. P -values were calculated using t

Supportive validation

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Experimental details: FIGURE 8: Proteomics identifies unique proteins recruited to paxillin in the integrin adhesions during EMT. (a) BioID technique. A control BirA*-eGFP construct and a BirA*-paxillin-eGFP construct are stably expressed in MCF10A cells, which are then treated with TGFbeta to induce EMT. BirA*-paxillin-GFP localizes to FAs and biotinylates proteins adjacent to paxillin when biotin is added to the medium. Biotinylated proteins are isolated and identified by mass spectrometry. (b) Normalized spectral counts for the consensus adhesome were clustered on the basis of an uncentered Pearson correlation using Cluster 3.0 and visualized using Java TreeView. From a subgroup of known paxillin interactors, we identify GIT2, LIMS1 (PINCH), and vinculin as enriched in TGFbeta-treated cells. Black indicates no protein detected in the +TGFbeta:-TGFbeta scale bar, and gray indicates no enrichment relative to the control. (c) Model for potential paxillin associations altered in response to TGFbeta treatment. PINCH, GIT2, and vinculin localize adjacent to paxillin, and these associations are enriched in TGFbeta-treated cells. (d-f) Immunofluorescence images of paxillin (green) and ILK (magenta). ILK is enriched in paxillin-rich adhesions in (d) MCF10A cells treated with TGFbeta and (e) PyMT cells treated with TGFbeta. Scale bar, 10 um (main image), 5 um (inset). (f) Immunofluorescence images of paxillin and ILK in PyMT cells on stiff (6 kPa) and soft (0.4 kPa) PA gels coated with fibronectin, showi

Supportive validation

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Experimental details: Figure 5 EGFR/AKT/mTOR and beta-catenin signaling in glioblastoma cells after treated with nanoparticles. Notes: ( A ) Confocal microscope images of U87 and U118 cells actin cytoskeleton. Cells were grown on extracellular matrix for 24 h and treated with diamond nanoparticles, graphite nanoparticles, or graphene oxide nanoparticles at a concentration of 20 mug/mL and incubated for 24 h. F-Actin was stained with phalloidin conjugated with Atto 633. ( B ) Western blot analysis of N-cadherin, pan-cadherin, vinculin, p-EGFR, and EGFR. GAPDH was used as a loading control. ( C ) Western blot analysis of nuclear and cytoplasmic protein fractions used for determination of beta-catenin protein level. PCNA and beta-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. ( D ) ELISA analysis of AKT and mTOR phosphorylation in comparison to control. Treatment with nanoparticles significantly reduced phospho-AKT ( P

Supportive validation

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Experimental details: Figure 9 Characterization of adipogenic phenotype of HFDPC and ADSC cultured in RAD16-I scaffold for 21 days. ( a ) FABP4 immunofluorescence of 3D constructs under adipogenic induction; ( b ) Nile red staining of 3D constructs under adipogenic induction; ( c ) FABP4 western blot of HFDPC and ADSC cultured in 2D cultures and in 3D RAD16-I scaffold under control and adipogenic medium. Vinculin was used as an internal control. The image is representative of three replicates.

Supportive validation

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Experimental details: Figure 1 CK2 is overexpressed in T-ALL compared to normal mononuclear cells . ( A ) Schematic showing PIK3CD and PIKFYVE genes in the PI3K pathway. ( B ) Baseline protein levels of CK2alpha, pCK2, and IKAROS in the T cell leukemia cell panel (CCRF-CEM, MOLT4, and primary T-ALL cells (labeled T-ALL#1)) were measured by Western blot and compared to peripheral blood mononuclear cells (MNCs). The protein level is graphed relative to vinculin as a loading control. ( C ) Radio-immunoblot showing phospho-IKAROS in a leukemia cell panel (CCRF-CEM, MOLT4, and primary T-ALL cells (labeled ALL#1-5)) compared to MNCs. ( D ) Radio-immunoblot showing a decrease in the phospho-IKAROS level following CX-4945 treatment. MOLT4 cells were treated with 10 muM of CX-4945 for 24 h. EV, endosomal vesicle; PIK3CD, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta; and PIKFYVE, phosphoinositide kinase, FYVE-type zinc finger containing; PI3K, phosphoinositide 3-kinase; P, phospho; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PDK1, phosphoinositide-dependent kinase; PI3P, phosphatidylinositol-3-phosphate; PI(3,5)P2, phosphotydile inositol 3,5-biphosphate.

Supportive validation

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Experimental details: Figure 4 CK2 inhibition restores IKAROS DNA binding and repression of PIK3CD and PIKFYVE . MOLT4, CEM, and T-ALL#1 cells were treated with 10 muM of CX-4945 for 24 h. IKAROS binding to the ( A ) PIK3CD and ( B ) PIKFYVE promoter region was confirmed using qChIP assay in vehicle- and CX-4945-treated cells. Results are mean +- SD of triplicates representative of one of three independent experiments. Molecular inhibition of CK2alpha in CEM and MOLT4 cells was achieved using shRNA. Two of four shRNAs showed a significant and similar decrease in CK2alpha. qRT-PCR shows the mRNA level of ( C ) CK2alpha and ( D ) PIK3CD and PIKFYVE in CK2-silenced MOLT4 (left panel) and CEM (right panel) cells. CEM and MOLT4 cells were treated with 10 and 20 muM of CX-4945 for 48 h, and T-ALL#1 primary leukemia cells were treated with 10 muM of CX-4945 for 12 h. mRNA and protein were extracted. ( E ) The mRNA level of PIK3CD and PIKFYVE was measured in CX-4945-treated CEM, MOLT4, and T-ALL#1 cells. ( F ) AKT and phosphorylated-AKT (p-AKT) protein levels were measured by Western blot. The protein level is expressed relative to vinculin. The p -value summaries are as follows: p

Supportive validation

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Experimental details: Fig. 2 EIF6 somatic missense mutations alter EIF6 protein stability or function to improve cell fitness. a Types of somatic EIF6 mutations. b Number and location of EIF6 mutations according to variant type. c Impact on the calculated energy of the folded state (DeltaDelta G mutation ) of EIF6 missense mutations. Mutant residue colored according to DeltaDelta G value. d DeltaDelta G mutation of 12 EIF6 missense mutations located at the RPL23-binding interface versus 85 other EIF6 missense mutations located in the remainder of EIF6 . Boxes center around the median and span the 25th and 75th percentiles, whiskers extend to the 10th and 90th percentiles. p value calculated using unpaired two tailed t -test. e Relative levels of EIF6 mRNA using a V5-specific qPCR primer (top panel) and V5 immunoblot from K562 cells 48 h after doxycycline treatment (bottom panel). Data shown is representative of three independent experiments. f Left panel: In silico modeling of EIF6-R96W. Right panel: V5 and VCL immunoblots of K562 cells with inducible EIF6-R96W versus V5-wild type EIF6 48 h after doxycycline treatment. Data shown is representative of three independent experiments. g Left panel: Change in the energy of binding (DeltaDelta G bind ) of missense mutations at RPL23 interface. Mutant residues are colored according to DeltaDelta G bind . Right panel: In silico modeling of EIF6 N106S mutation. h V5, EIF6, and RPL3 immunoblots of sucrose gradient fractions from polysome profiles of doxycyc

Supportive validation

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Experimental details: Figure 3 Lamellipodia and stress fiber formation are increased in A375 cells expressing GFP-PMCA4b but not in those expressing GFP-PMCA4b-LA. ( A ) Confocal microscopy images of A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells labeled with Phalloidin-TRITC. Arrowheads show stress fibers. Scale bar, 20 um. The fractions of stress fiber-positive cells in A375-GFP ( n = 55), A375-GFP-PMCA4b-LA ( n = 72), A375-GFP-PMCA4b ( n = 60) +- PMCA4b siRNA ( n = 68), and negative siRNA ( n = 49) cultures were determined. siRNA confocal microscopy images are presented in Figure S3A . Relative abundance of cells with stress fibers is indicated by the bar graph. Confocal sections were taken from the bottom of the cells to show the stress fibers. ( B ) Confocal microscopy images of A375 and A375-GFP-PMCA4b cells immunostained with vinculin and labeled with Phalloidin-TRITC. Scale bar, 20 um. Insets show part of the cells with higher magnification. Arrowheads show the differential localization of vinculin. Scale bar, 5 um. ( C ) A375 and A375-GFP-PMCA4b cells were cultured in a 6-well plate for 48 h. Vinculin protein expression from total cell lysate was analyzed by Western blotting. beta-tubulin was used as a loading control. Data represent mean +- S.E.M from three independent experiments (*** p < 0.001).

Supportive validation

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Experimental details: Immunofluorescence analysis of signal mechanotransduction proteins. Actin (blue) beta1-integrin (green), pFAK Y397 (red), pFAK Y861 (red) and vinculin (red) staining in PANC-1 (left) and MiaPaCa-2 (right) cells in ( a ) 0.15% RAD16-I; ( b ) 0.5% RAD16-I and ( c ) 2D cultures. Merge of green and red channels is also shown. Scale bars represent 10 um; ( d ) Mander's colocalization coefficients of PANC-1 and MiaPaCa-2 cells (n = 5). Statistical differences are indicated as * for p value < 0.05, ** for p value < 0.01 and *** for p value < 0.001.

Supportive validation

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Experimental details: Figure 6 ( A ) Immunoblots of FAK-related signaling molecules. The figures shown are representative of one experiment. ( B ) The bar graphs show densitometric data (mean+-SD) from three independent experiments. Note: * P

Supportive validation

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Experimental details: Figure 3 Propidium iodide (PI) 488 assay analysis. Notes: Effect of C 60 on the number (percentage) of ( A ) HS-5, ( B ) HepG2 or ( C ) C3A cells in the cycle phases. ( D ) Relative HS-5, HepG2 and C3A cell population in G2/M cycle (%). ( E ) Western blot analysis of beta-catenin, N-cadherin, vinculin and PCNA. GAPDH was used as a loading control. Abbreviations: C 60 , fullerenes; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen.

Supportive validation

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Experimental details: Fig. 7 Galphaq overexpression enhances cAMP response of endogenous beta 2 -AR. a cAMP accumulation in HEK293T cells either overexpressing Galphaq (mCer-tag) or an equivalent number of untransfected control cells following stimulation with increasing concentration of fenoterol (0.3 nM to 100 muM) in the presence of 2 muM forskolin (see Methods). The increase in cAMP with fenoterol concentration was fit to a four-parameter logistic function to estimate EC 50 and E max (Methods). Values are mean +- SD from six experiments with at least three repeats per experiment ( n >= 6). Statistically significant differences in the EC 50 and E max values were assessed by Students' t -test. b cAMP accumulation in HEK293T cells either overexpressing Galphaq (mCer-tag) or equivalent number of untransfected control cells, following stimulation with fenoterol (10 muM) and forskolin (2 muM), in the presence of PKC inhibitors Bim1 (1 muM) or Go6983 (1 muM), or DMSO. Cells overexpressing Galphaq had accumulated greater cAMP even in the presence of PKC inhibitors. The drug treatments do not influence the cAMP response across the three untransfected or the three transfected conditions. Box-and-whisker plots: center line is median, box ends are upper and lower quartiles, whisker ends are 1.5 x IQR (interquartile range) from four independent experiments with three technical replicates per experiment ( n >= 4). Statistically significant differences were assessed by two-way ANOVA followed by Tukey's post-

Supportive validation

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Experimental details: Fig. 2 Quality controls for PLA in mouse tissue using primary antibodies raised in mouse. During PLA assay on mouse liver tissue samples from SFGR infection mouse on day 19, there is no specific signals if no primary antibody employed (only normal mouse and rabbit IgGs) ( a ), using rabbit anti-vinculin antibodies and normal mouse IgG employed ( b ), using rabbit anti-vinculin antibodies and mouse anti-talin, but no oligonucleotide probes ( e ). There is specific signal detected when rabbit anti-vinculin and mouse anti-talin antibodies were employed during PLA on mouse liver tissue ( c , d ). Nuclei were counter-stained with DAPI (blue). Scale bars indicate 20 um.