Cells were seeded on glass coverslips, fixed with 4% PFA (Sigma-Aldrich) and incubated overnight at 4°C with the following primary antibodies: anti-PanCytokeratin (Mouse 1:200, Dako, Glostrup, Denmark), anti-Epcam (Mouse 1:1000, clone HEA-125, GeneTex, Irvine, CA, USA), anti-AQ-1 (Mouse 1:50, clone B-11, Santa Cruz Biotechnology, Heidelberg, Germany), anti-CD13-PE (Mouse 1:25, Biolegend, San Diego, CA, USA), anti-CD13-FITC (Mouse 1:25, Abcam, Cambridge, UK), anti-N-cadherin (Rabbit 1:50, Abcam, and Mouse 1:50, clone 32/N, Becton Dickinson, San Josè, CA, USA), anti-Calbindin (Mouse 1:100, clone CB-955, Sigma-Aldrich), anti-E-cadherin (Mouse 1:50, Becton Dickinson, and Rabbit 1:50, Cell Signalling Technology, Danvers, MA, USA) and anti-Paxillin (Mouse 1:50, Becton Dickinson). When necessary, the secondary antibodies Alexa 488 conjugated anti-mouse IgG and Alexa 594 conjugated anti-rabbit IgG (1:100, Molecular Probes, Carlsberg, CA, USA) were used. Stress fibers were labeled by Alexa-Fluor-594-phalloidin (1:100, Molecular Probes) and nuclei counterstained with Mounting DAPI (Molecular Probes). Immunofluorescence images were obtained with a Zeiss LSM810 confocal microscope, using a 63x objective, equipped with Zen2009 software (Zeiss, Oberkochen, Germany).
Anti paxillin
Manufactured by BD
Sourced in United States
Anti-paxillin is a laboratory reagent used for the detection and analysis of the paxillin protein in cell samples. Paxillin is a focal adhesion-associated protein that plays a role in the regulation of cell adhesion and migration. The anti-paxillin reagent can be used in various cell biology research applications, such as immunofluorescence microscopy and Western blotting, to visualize and quantify the expression and localization of paxillin within cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Anti actin, by Merck Group (4 mentions)
Anti vinculin, by Merck Group (4 mentions)
Anti e cadherin, by BD (3 mentions)
Dasatinib, by Bristol-Myers Squibb (3 mentions)
Anti fak, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti p130cas, by BD (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti psrc y416, by Cell Signaling Technology (2 mentions)
Anti gm130, by BD (2 mentions)
Anti ppaxillin y118, by Cell Signaling Technology (2 mentions)
Anti src clone 36d10, by Cell Signaling Technology (2 mentions)
Phalloidin tritc, by Thermo Fisher Scientific (2 mentions)
Anti lc3b, by Cell Signaling Technology (2 mentions)
39 protocols using anti paxillin
1
Immunofluorescence Analysis of Cell Markers
2
Recombinant Cytokine and Antibody Protocol
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Recombinant TNFα, CX3CL1 and anti-CX3CL1 were obtained from R&D Systems Europe Ltd (Abingdon, United Kingdom). Anti-phospho-paxillin (Y118) was obtained from Invitrogen (Carlsbad, CA); Anti-paxillin from Becton Dickinson (San Jose, CA); Anti-phospho-Akt (Ser473) and anti-phospho-ERK-1/2 (Thr202 and Tyr204) were purchased from New England Biolabs (Beverly, MA). Anti-ERK1/2, anti-Akt, anti-AS160 and anti-phospho-(Ser/Thr)-AS160 were obtained from Cell Signaling Technology (Beverly, MA). Anti-p65 subunit of NF-κB (C-20) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and monoclonal anti-actin from Sigma. Anti-IRS-1 and IRS-2 were a gift from Dr. M.F. White (Children's Hospital, Harvard University, Boston, MA). Anti-insulin and anti-glucagon were from Dako (Glostrup, Denmark).
3
Antibody Profiling for Focal Adhesion
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Antibodies used were as follows: anti-Paxillin (RRID:AB_647289 ), anti-GM130 (RRID:AB_398141 ), anti-p130Cas (RRID:AB_397667 ) and anti-RACK1 (RRID:AB_397577 ) antibodies (BD Transduction Laboratories, New Jersey, USA), anti-IFITM3 (RRID:AB_2122095 ) (Abcam, Cambridge, UK), anti-CoxIV (RRID:AB_10694213 ), anti-FAK (RRID:AB_10694068 ), anti-pFAK Y397 (RRID:AB_2173659 ), anti-pPaxillin Y118 (RRID:AB_2174466 ), anti-Rab7 (RRID:AB_1904103 ), anti-pSrc Y416 (RRID:AB_331697 ), anti-Src (clone 36D10) (RRID:AB_10693939 ), anti-LC3B (RRID:AB_2137707 ) and anti-GAPDH (RRID:AB_10622025 ) (Cell Signaling Technologies, Danvers, MA, USA), and anti-Dctn1 (RRID:AB_10842517 ), anti-Ambra1 (RRID:AB_2636939 ) and anti-pSrc Y416 (RRID:AB_309898 ) antibodies and anti-FAK (clone 4.47)-conjugated agarose antibody (RRID:AB_310789 ) (Millipore, Billerica, MA, USA). LC3B antibody was from MBL (RRID:AB_1279144 ) (MBL International, Woburn, MA, USA). TRITC-phalloidin was purchased from Life Technologies (RRID:AB_2572408 ) (Paisley, UK). Anti-rabbit (RRID:AB_2099233 ) or mouse (RRID:AB_330924 ) peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ, USA) and PF562271 from Pfizer (Groton, CT, USA).
4
Integrin-Mediated Cellular Adhesion and Apoptosis
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Either LN111 or LN121 (both 10 μg ml–1, Biolamina) was loaded on coverglass slips precoated with PLL (Sigma), and SUM159-LM1 cells were then seeded onto the coverslips. To inhibit integrin β1 function, anti-integrin β1 blocking antibody (2.5 μg ml–1, Merck) was used. After 24–48 h, cells were fixed with 2% formaldehyde/PBS on ice, permeabilized with 0.1% Triton X-100 and 0.1% Tween20, blocked and incubated with either anti-paxillin (1:1,000, BD Transduction Laboratories) or anti-cleaved caspase 3 (1:250, Cell signaling). Cy3- or Alexa-488-conjugated secondary antibodies (1:500, Invitrogen) were used to reveal staining and DAPI (BioLegend) to stain nuclei. Cells were imaged using a Cell observer microscope (Zeiss), and cell area and number of apoptotic cells per field were analyzed with FIJI (ImageJ).
5
Immunohistochemistry and Flow Cytometry Protocols
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We used the following primary antibodies: anti-FN (1:1,000, Millipore, Burlington MA USA), anti-α-SMA-Cy3 conjugated (1:500, Sigma-Aldrich, St. Louis, MO USA), anti-paxillin (1:300, BD Transduction, Spain), anti-keratin 6 (1:500, Covance, Spain), anti p-Smad2/3 (1:300, Thermo Fisher, Spain), anti-α5 integrin (1:100, Cell Signaling Technology, The Netherlands) and anti-Ki67 (1:200, Abcam, Cambridge, UK). For flow cytometry, we used anti-α5 integrin (1:200 BD Pharmingen, Spain) and anti β3 integrin (1:200 BD Bioscience, Spain). Secondary antibodies: anti-rabbit Alexa Fluor 488 (1:400 Thermo Fisher, Spain) and anti-mouse Alexa Fluor 647 (1:500, Invitrogen). To stain the cytoskeleton, we used Rhodamine Phalloidin (1:500 Thermo Fisher). To stain nuclei, we used either DAPI (1:10,000, Thermo Fisher) or Hoechst (1:10,000, Thermo Fisher).
6
Immunoblotting for Cellular Signaling
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anti Fyn (sc-16), and anti phospho-paxillin (sc-101774; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti - and anti general-Akt (#9721 and #2938, respectively; Cell signaling Technology, Danvers, MA, USA). Anti FAK (#AHO0502; Biosource International (Camarillo, CA, USA). Anti phospho-FAK (#44625G; Invitrogen, Carlsbad, CA, USA). Anti actin (#MAB1501; Millipore, Temecula, CA, USA). Anti paxillin (#610052; BD Transduction Laboratories, San Diego, CA, USA). Rodhamine Phalloidin (Molecular Probes, Eugene, OR,USA)
7
Immunostaining of Cytoskeletal Proteins
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The following tubulin antibodies were used: monoclonal anti-α-tubulin (Clone DM 1A) and anti-acetylated α-tubulin (Clone 6-11B-1) (Sigma-Aldrich), monoclonal anti-tyrosinated α-tubulin (YL1/2, Santa Cruz), and polyclonal anti-detyrosinated α-tubulin (Millipore). The following polyclonal antibodies were used: anti-GAPDH (HyTest), anti-Kif5A (Abcam), and anti-TTL (Proteintech Group). The following monoclonal antibodies were used: anti-β-catenin (Sigma-Aldrich), anti-KANK1 (Invitrogen), anti-paxillin (BD Transduction Laboratories), anti-sc35 (Abcam), and anti-vinculin (Sigma-Aldrich). The monoclonal antibody directed against sucrase-isomaltase (SI) (DRBB2/158) was generously provided by A. Quaroni. The plasmid mCherry-Vinculin-N-21 was a gift from Michael Davidson (Addgene plasmid #55160; RRID:Addgene_55160 ).
8
Protein Interactions in Cell Signaling
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Antibodies used were as follows: anti-Eps8, anti-paxillin, anti-GM130 and anti-p130Cas antibodies (BD Transduction Laboratories, NJ), anti-FAK, anti-pSrc Y416, anti-Src (clone 36D10), anti-LC3B, anti-GAPDH, anti-pPaxillin Y118 and anti-β-actin antibodies (Cell Signaling Technologies, Danvers, MA), as well as anti-FAK antibody conjugated to agarose (clone 4.47) (Millipore, Billerica, MA) and anti-LC3B for immunoprecipitations (MBL, Woburn, MA). The anti-TagCGYFP antibody was purchased from Evrogen (Cambridge, UK). TRITC–phalloidin was purchased from Life Technologies (Paisley, UK). Horseradish-peroxidase-conjugated secondary antibodies against rabbit or mouse IgG were purchased from Cell Signaling Technologies. Dasatinib was obtained from Bristol Myers Squibb (Princeton, NJ). The pEGFP-Eps8 1–821 and pEGFP-Eps8 1–535 constructs were a generous gift from Giorgio Scita (FIRC Institute of Molecular Oncology, Milan, Italy).
9
Src-Akt-mTOR Signaling Pathway Monitoring
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Antibodies used were: anti-p-Src Y416, anti-Src 36D10, anti-LC3B, anti-FAK, anti-p-PDK1 S241, anti-PDK1, anti-p-Akt S473, anti-Akt, anti-GAPDH, anti-p-mTOR S2448, anti-p-mTOR S2481, anti-mTOR, anti-p-S6 S235/236, anti-p-S6 S240/244, anti-S6, anti-p-p70S6K T389, anti-p70S6K, anti-mouse and anti-rabbit IgG-peroxidase conjugated secondary antibodies (all New England Biolabs, Herts, UK), anti-LC3 (MBL, MA, USA), and anti-paxillin (BD Transduction Laboratories, Oxford, UK). Alexa 488 and Alexa 594 conjugated secondary antibodies, Deep Red Cell Mask and Vectashield microscope mounting medium with DAPI were from Invitrogen (Paisley, UK).
Tocriscreen Kinase Inhibitor Toolbox, SB218078, SB216763, ZM306416, PD407824, BIO, Ki8751 and PF4708671 were all from R & D (Abington, UK), hygromycin B from Merck Biosciences (Nottingham, UK), dasatinib from Bristol Myers Squibb (Middlesex, UK) and 3MA from Sigma (Poole, UK). The Micro BCA Protein Assay Kit was from Pierce Ltd. (Northumbria, UK), Lipofectamine2000 from Invitrogen (Paisley, UK) and HiPerFect from Qiagen (Crawley, UK). siRNA was from Dharmacon (Colorado, USA).
Tocriscreen Kinase Inhibitor Toolbox, SB218078, SB216763, ZM306416, PD407824, BIO, Ki8751 and PF4708671 were all from R & D (Abington, UK), hygromycin B from Merck Biosciences (Nottingham, UK), dasatinib from Bristol Myers Squibb (Middlesex, UK) and 3MA from Sigma (Poole, UK). The Micro BCA Protein Assay Kit was from Pierce Ltd. (Northumbria, UK), Lipofectamine2000 from Invitrogen (Paisley, UK) and HiPerFect from Qiagen (Crawley, UK). siRNA was from Dharmacon (Colorado, USA).
10
Antibody Panel for Autophagy and Cytoskeleton Regulation
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Anti-Atg5 and anti-LARG antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Anti-paxillin, anti-p62, anti-actin, and anti-tubulin antibodies were purchased from BD Biosciences (San Jose, CA), MBL (Aichi, Japan), Millipore (Billerica, MA), and Invitrogen (Carlsbad, CA), respectively. Anti-GEF-H1, anti-RhoB, and anti-RhoC antibodies were from Cell Signaling Technologies (Danvers, MA). Anti-RhoA and anti-Rac1 antibodies were from Cytoskeleton Inc. (Denver, CO). Anti-LC3, anti-phosho FAK and anti-GAPDH were purcharsed from NanoTools (Teningen, Germany), Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Bafilomycin A1 was obtained from Sigma-Aldrich. Rho inhibitor 1 was obtained from Cytoskeleton Inc. and Chemdea LLC. (Ridgewood, NJ), respectively. All other chemicals were from Wako Co. (Osaka, Japan).
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