Mouse anti vinculin

Manufactured by Santa Cruz Biotechnology

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Mouse anti-vinculin is a primary antibody that specifically recognizes the vinculin protein. Vinculin is a cytoplasmic protein involved in the linkage of integrin adhesion molecules to the actin cytoskeleton, and is important for cell-cell and cell-matrix adhesion.

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Vinculin (138 citations) Anti-vinculin (69 citations) Mouse monoclonal anti-vinculin (4 citations) Mouse monoclonal anti-vinculin (7f9), (3 citations) Mouse anti-vinculin antibody (3 citations)

12 protocols using mouse anti vinculin

Western Blot Analysis of Stem Cell Markers

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Undifferentiated and differentiated ESCs were lysed in radioimmunoprecipitation assay buffer containing 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, and protease inhibitor cocktail (Sigma–Aldrich), and analyzed by Western blot. The following primary antibodies were used: rabbit anti-Yap (1:1000; D8H1X; Cell Signaling Technology), rabbit anti-Dnmt3l (1:1000; E1Y7Q; Cell Signaling Technology), mouse anti-Vinculin (1:1000; G11; Santa Cruz Biotechnology), and mouse anti-Flag (1:2000; Sigma). Western blots were developed with an ECL system (BioRad) using the following horseradish peroxidase–conjugated antibodies: anti-rabbit IgG (1:10,000), antimouse IgG (1:5000; both from Amersham Pharmacia Biotech).

Passaro F., De Martino I., Zambelli F., Di Benedetto G., Barbato M., D’Erchia A.M., Manzari C., Pesole G., Mutarelli M., Cacchiarelli D., Antonini D., Parisi S, & Russo T. (2020). YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation. The Journal of Biological Chemistry, 296, 100138.

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Western Blotting of EMT Markers

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Western blotting was performed as described previously using whole cell lysates [14 (link)]. Cells were harvested to prepare cell lysates two weeks after initiating of mutant KRAS^V12 induction. Primary antibodies used were mouse anti-E-cadherin (Cat#610181, BD Biosciences), mouse anti-Vimentin Cat#550513, BD Biosciences), mouse anti-p53 (Cat#sc-126, Santa Cruz, USA), mouse anti-p21 Waf/Cip/CDKN1A (Cat#sc-6246, Santa Cruz), mouse anti-KRAS (Cat#sc-30, Santa Cruz), rabbit anti-AKT (pan) (Cat#4691, Cell Signaling Technology), rabbit anti-phospho-AKT (ser 473) (Cat#4060, Cell Signaling Technology), rabbit anti-p44/42MAPK(Erk1/2) (Cat#9102, Cell Signaling Technology), rabbit anti-phospho-p44/42MAPK(Erk1/2) (Cat#4370, Cell Signaling Technology), mouse anti-Actin (Cat#A2228, Sigma-Aldrich), mouse anti-Vinculin (Cat#SC-73614, Santa Cruz), rabbit anti-α-tubulin (Cat# PM054-7, MBL Life Science, USA), and rabbit anti-phospho-p53 (Ser 15) (Cat#9284, Cell Signaling Technology) antibodies. Actin, Vinculin, or α-tubulin protein levels were used as a control for the adequacy of equal protein loading. Anti-rabbit or anti-mouse (GE Healthcare, Buckinghamshire, England) antibodies were used at 1:2000 dilution as secondary antibodies.

Bisgaard H, & Møller H. (1999). Changes in risk of hospital readmission among asthmatic children in Denmark, 1978-93. BMJ : British Medical Journal, 319(7204), 229-230.

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Western Blot Analysis of Protein Markers

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Protein extracts were mixed with 4× sample buffer (8% SDS, 20% v/v glycerol, 0.002% bromphenolblue, 62.5 mM Tris-Cl, pH 6.8) supplemented with 100 mM dithiothreitol (DTT) and incubated for 10 min at 95°C. Proteins were separated on 4–15% Criterion TGX precast gels (Bio-Rad) and transferred to nitrocellulose membranes using the Trans-Blot^® Turbo™ Transfer System (Bio-Rad). Membranes were blocked using 5% non-fat dry milk (Blotto, Santa Cruz Biotechnologies) in TBS, 0.1% v/v Tween® 20 (Merck) for 30 min to 1 h at room temperature. Primary antibody incubations were performed overnight at 4°C in blocking buffer. The following primary antibodies were used: rabbit anti-PTPA (Cell Signaling Technologies, 3330, 1:1000 dilution), mouse anti-PTPA (BioLegend, 824401, 1:1000 dilution), mouse anti-PP2A C (Sigma-Aldrich, 05-421, 1:1000 dilution), rabbit anti-LC3B (Novus, NB100-2220, 1:1000 dilution), rabbit anti-GADD34 (Proteintech, 10449-1-AP, 1:1000 dilution), mouse anti-vinculin (Santa Cruz Biotechnology, V284, 1:2000 dilution). After washing in TBS, 0.1% v/v Tween^® 20, blots were incubated for 1 h at room temperature with fluorescently conjugated Alexa Fluor Plus 800 donkey anti-mouse and 700 donkey anti-rabbit (both from ThermoFisher) secondary antibodies. After washing in TBS, 0.1% v/v Tween^® 20, blots were imaged and analysed using the Odyssey CLx imaging system (LI-COR Biosciences).

Fevga C., Tesson C., Carreras Mascaro A., Courtin T., van Coller R., Sakka S., Ferraro F., Farhat N., Bardien S., Damak M., Carr J., Ferrien M., Boumeester V., Hundscheid J., Grillenzoni N., Kessissoglou I.A., Kuipers D.J., Quadri M., Corvol J.C., Mhiri C., Hassan B.A., Breedveld G.J., Lesage S., Mandemakers W., Brice A, & Bonifati V. (2022). PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability. Brain, 146(4), 1496-1510.

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Western Blot Analysis of Cathepsin H

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Fibroblast pellets were lysed in RIPA buffer with a broad-spectrum protease inhibitor (cOmplete tablets; Roche). The protein content was determined using the DC protein assay (Bio-Rad) according to the manufacturer’s instructions. Equal amounts of protein (20–30 μg; diluted in 4× sample buffer [125 mmol/L Tris/HCl, pH 6.8; 4% sodium dodecyl sulfate, 2% β-mercaptoethanol, 20% glycerol, 1 mg bromophenol blue]) were heated at 95°C for 5 minutes and separated with electrophoresis using precast polyacrylamide gradient gels (NuPAGE 4%-12%, Bis-Tris; Thermo Fisher Scientific) under reducing conditions. Proteins were transferred to polyvinylidene difluoride membranes (Merck, Readington, NJ). Nonspecific binding was blocked in 5% milk powder in Tris-buffered saline containing 0.05% Tween-20 (Merck). Blots were incubated overnight with mouse anti–cathepsin H and mouse antivinculin (both from Santa Cruz Biotechnology, Santa Cruz, CA). Detection was performed by horseradish peroxidase–conjugated goat anti-mouse antibodies (Agilent Technologies) and chemiluminescence (Lumi-Light Western Blot Substrate; Roche) was used to visualize the target proteins.

Dang H., Harryvan T.J., Liao C.Y., Danen E.H., Spalburg V.N., Kielbasa S.M., Mei H., Goeman J.J., de Jonge-Muller E.S., Janson S.G., van der Reijden J.J., Crobach S., Hardwick J.C., Boonstra J.J., de Miranda N.F, & Hawinkels L.J. (2023). Cancer-Associated Fibroblasts Are Key Determinants of Cancer Cell Invasion in the Earliest Stage of Colorectal Cancer. Cellular and Molecular Gastroenterology and Hepatology, 16(1), 107-131.

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HeLa Cell Lysis and Western Blot

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HeLa cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris with pH 7.4, 1% Triton, 0.5% Nonidet P40, 10% glycerol, and 2.5% sodium deoxycholate) plus protease and phosphatase inhibitors (Roche Diagnostic, Germany, 11836153001). Protein concentrations were determined with the Bio-Rad Protein Assay Dye kit (Bio-Rad, United States, 5000006). Cell extracts were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore, Germany, Immobilon-P IPVH00010). Blocking was performed at room temperature in TBS 1X 0.1% Tween 5% low-fat milk for 1 h. Membranes were incubated with primary antibodies overnight at +4°C, followed by incubation with horseradish peroxidase-conjugate secondary antibodies (Bio-Rad) and revealed with western chemoluminescent HRP substrate (Millipore, Immobilon WBKLS0500). Chemoluminescent signals were acquired with the iBright CL1000 Imaging System (Thermo Fisher, United States). Quantitative analysis was performed using ImageJ software. Primary antibodies used were rabbit anti-p62 (MBL International Corporation, United States, PM045), mouse anti-COXII (Abcam, United Kingdom, ab110258), mouse anti-COXIV (Abcam, United Kingdom, ab33985), rabbit anti-LC3 A/B (Cell Signaling, United States, D3U4C, 12741S), rabbit anti-HSP90 (Cell Signalling; United States, E289, 4875), and mouse anti-vinculin (Santa Cruz Biotechnology, United States, 7F9, sc-73614).

Schiavi A., Runci A., Maiorino T., Naso F.D., Barenys M., Fritsche E., Strappazzon F, & Ventura N. (2022). Cobalt chloride has beneficial effects across species through a hormetic mechanism. Frontiers in Cell and Developmental Biology, 10, 986835.

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Western Blot Analysis of Mitochondrial Proteins

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Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris with pH 7.4, 1% Triton, 0.5% Nonidet P40, 10% glycerol, and 2.5% sodium deoxycholate) supplemented with protease inhibitors (Roche Diagnostics, Basel, Switzerland, 11836153001). Proteins were resolved by SDS PAGE and transferred on a PVDF membranes (Millipore/MERCK, Darmstadt, Germany, Immobilon-P IPVH00010). About 20–30 μg of extract per lane were loaded. Blocking and HRP-conjugated secondary antibodies (Biorad) incubations were performed at room temperature in TBS containing 0.1% Tween and 5% low fat milk or 1 h. Fluorescent signals were revealed using chemo-luminescent HRP substrate (Millipore, Immobilon, WBKLS0500). Primary antibody incubations were carried out overnight at +4 °C. Antibodies used were: rabbit anti-β actin (Sigma Aldrich, A2066), mouse anti-COXII (Abcam, Cambridge, United Kingdom, ab110258), rabbit anti-PINK1 (Novus Biologicals, Centennial, Colorado, USA, BC100-494), mouse anti-GAPDH (Sigma Aldrich, SAB1405848), rabbit anti-Parkin (Cell Signalling Technologies, Danvers, Massachusetts, USA, 2132), mouse anti-COXIV (Abcam, ab33985), mouse anti-vinculin (Santa Cruz Biotechnology, Dallas, Texas, USA, 7F9, sc-73614), mouse anti-TOMM20 (Santa Cruz Biotechnology, sc-17764), rabbit anti-PARP (Cell Signalling Technologies, 9542).

Naso F.D., Bruqi K., Manzini V., Chiurchiù V., D’Onofrio M., Arisi I, & Strappazzon F. (2024). miR-218-5p and doxorubicin combination enhances anticancer activity in breast cancer cells through Parkin-dependent mitophagy inhibition. Cell Death Discovery, 10, 149.

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Optimizing YTHDC2 Antibody Selection for CLIP and Immunoblotting

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Primary antibodies used for CLIP were rabbit anti-YTHDC2 (Abcam ab220160) and rabbit anti-YTHDC2 (Proteintech 27779-1-AP). We also tested the following antibodies and did not include them in our CLIP protocol due to poor performance: rabbit anti-YTHDC2 (Bethyl Laboratories A303-026A), rabbit anti-YTHDC2 (Bethyl Laboratories A303-025A), and rabbit anti-YTHDC2 (Invitrogen PA5-57920). The following antibodies were used for immunoblots: rabbit anti-YTHDC2 (1:5000; Abcam ab220160), rabbit anti-YTHDC2 (1:1000; Abcam ab176846), mouse anti-β-Actin (1:20,000; Abcam ab6276), and mouse anti-Vinculin (1:500; Santa Cruz Biotechnology sc-73614). Primary antibodies used for histology were 1 µg/mL mouse anti-SYCP3 (Santa Cruz Biotechnology sc-74569), 0.2 µg/mL rabbit anti-γH2AX (Abcam ab11174), and 1 µg/mL rabbit anti-pH3 (Millipore 06-570).

Saito Y., Hawley B.R., Puno M.R., Sarathy S.N., Lima C.D., Jaffrey S.R., Darnell R.B., Keeney S, & Jain D. (2022). YTHDC2 control of gametogenesis requires helicase activity but not m6A binding. Genes & Development, 36(3-4), 180-194.

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Western Blot Protein Detection Protocol

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Proteins were separated on 4–12% gradient NuPAGE Novex Bis-Tris gel (Life Technologies), transferred to PVDF membrane (Millipore), blocked in 1 × TBS-Tween with 5% non-fat dry milk and incubated with corresponding primary antibody and secondary antibodies. The following antibodies were used: rabbit anti-TGM2, (1:1,000; Cat. No. HPA021019, Sigma-Aldrich, Prestige Antibodies Powered by Atlas Antibodies), Rabbit anti-CLIC3 (1:3,000; produced in house31 (link)), Rabbit anti-β-tubulin (1:1,000; Cat. No. sc-9104, Santa Cruz), Mouse anti-Vinculin (1:1,000; Cat. No. V9131, Sigma-Aldrich) and Mouse anti-αSMA (1:1,000; Cat. No. A5228, Sigma). As secondary antibodies, horseradish peroxidase-conjugated (1:10,000; Cat. No. HAF008 and HAF007, H&D systems,), IRDye 680RD (1:10,000; Cat. No. 926-68072, LI-COR) and IRDye 800CW (1:10,000; Cat. No. 926-32213, LI-COR) were used. Images were captured with a Bio-Rad GS-800 Calibrated densitometer (Quantity-One software version 4.6.3) or a LI-COR Odyssey CLx scanner (Image Studio software, version 5.0.21) for chemluminiscent or fluorescence western blots, respectively. Representative images from reproducible, independent experiments are shown. Uncropped scans of the western blots are reported in Supplementary Information (Supplementary Figs 9 and 10).

Hernandez-Fernaud J.R., Ruengeler E., Casazza A., Neilson L.J., Pulleine E., Santi A., Ismail S., Lilla S., Dhayade S., MacPherson I.R., McNeish I., Ennis D., Ali H., Kugeratski F.G., Al Khamici H., van den Biggelaar M., van den Berghe P.V., Cloix C., McDonald L., Millan D., Hoyle A., Kuchnio A., Carmeliet P., Valenzuela S.M., Blyth K., Yin H., Mazzone M., Norman J.C, & Zanivan S. (2017). Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity. Nature Communications, 8, 14206.

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Immunoprecipitation and Western Blot Analysis

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Rabbit-anti-ID3 (Cell Signaling, Cat#9837) and for IP of endogenous ID3 we used agarose beads conjugated with mouse-anti-ID3(Santa Cruz, Cat#sc-56712). Mouse-anti-beta-Actin (Santa Cruz, Cat#sc-47778). Mouse-anti-Vinculin (Santa Cruz, Cat#sc-25336). Mouse-anti-phospho-Histone H2A.X (S139) (Merck, Cat#05-636). Rabbit-anti-phospho-Histone H2A.X (S139) (Abcam, Cat# ab2893). Rabbit-anti-RAD51 (Calbiochem, Cat#PC130). Rabbit-anti-RAD51 (Abcam, Cat#ab176458). Rabbit-anti-XRCC4 (GenTex, Cat#GTX109632). Rabbit-anti-phospho-RPA (S4/S8) (Bethyl, Cat#A300-245A). Mouse-anti-Histone H2B (Abcam, Cat#ab52484). Rabbit-anti-DNA-PKsc (Cell Signaling, Cat#4602). Rabbit-anti-CtIP (Abcam, Cat#70163). Goat-anti-MDC1 (Santa Cruz, Cat#sc-27737). Rabbit-anti-MDC1 (Abcam, Cat#ab11169). Rabbit-anti-NBS1 (Novus Biologicals, Cat#NB100-143). Mouse-anti-RAD50 (Abcam, Cat#ab89). Rabbit-anti-RECQL (Abcam, Cat#ab151501). Mouse-anti-RPA32 (Santa Cruz, Cat#sc-53496). Mouse-anti-Flag-HRP (Sigma, Cat#A8592). Mouse-anti-IgG (Santa Cruz, Cat#sc-2025). Mouse-anti-CenpF (BD bioscience, Cat#610768). Goat-anti-mouse IgG-HRP (Cell Signaling, Cat#7076P2). Goat-anti-rabbit IgG-HRP (Cell Signaling, Cat#7074S). Goat-anti-mouse IgG-AlexaFluor 594 (Molecular Probes, Cat#A11005). Goat-anti-rabbit IgG-AlexaFluor 488 (Molecular Probes, Cat#A11008).

Bakr A., Hey J., Sigismondo G., Liu C.S., Sadik A., Goyal A., Cross A., Iyer R.L., Müller P., Trauernicht M., Breuer K., Lutsik P., Opitz C.A., Krijgsveld J., Weichenhan D., Plass C., Popanda O, & Schmezer P. (2021). ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition. Nucleic Acids Research, 49(20), 11666-11689.

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Antibody Characterization for EGFR Signaling

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The primary antibodies used for immunoblot analysis and immunohistochemical staining were rabbit anti-EGFR, mouse anti-vinculin, mouse anti-Tom23, goat anti-Tim23, rabbit anti-Mfn1, mouse anti-Drp1 (all above from Santa Cruz Biotechnology, CA, USA), mouse anti-MTCO1 (Abcam, Cambridge, UK), rabbit anti-phospho-EGFR (Tyr1068), rabbit anti- phospho-Drp1 (Ser637) (all above from Cell Signaling Technology, Inc., MA, USA), mouse anti-OPA1 (BD Biosciences, NJ, USA) and mouse anti-myc, mouse anti-Flag, mouse anti-β actin antibody (all above from Sigma-Aldrich, Inc., MO, USA). The primary antibodies used for immnofluorescence staining were rabbit anti-EGFR (Abcam, Cambridge, UK) antibody.

Che T.F., Lin C.W., Wu Y.Y., Chen Y.J., Han C.L., Chang Y.L., Wu C.T., Hsiao T.H., Hong T.M, & Yang P.C. (2015). Mitochondrial translocation of EGFR regulates mitochondria dynamics and promotes metastasis in NSCLC. Oncotarget, 6(35), 37349-37366.

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