Mouse anti paxillin

Manufactured by BD

Sourced in United States

Mouse anti-paxillin is a monoclonal antibody that recognizes the paxillin protein, which is a key structural and signaling component of focal adhesions in cells. This antibody can be used to detect and study the localization and expression of paxillin in various cell and tissue types.

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29 protocols using mouse anti paxillin

Antibody Profiling for Ephrin-Eph Signaling

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Antibodies used in this study include rabbit anti-ephrinB2 (PA5-35062; Thermo Fisher Scientific), mouse anti-EphB4 (H-10) (sc-365510; Santa Cruz Biotechnology), mouse anti-flag (F3165; Sigma-Aldrich), goat anti-DsRed (sc-33354; Santa Cruz Biotechnology), mouse monoclonal anti-p-Tyr (sc-508; Santa Cruz Biotechnology), mouse anti-paxillin (610051; BD Transduction Laboratories), and anti-α-tubulin (T6074; Sigma-Aldrich). Mouse anti-HA (12CA5) and rabbit anti-EGFP antibodies were developed in the laboratory. Rabbit anti-HA (923501) and anti-DYKDDDDK Tag (L5) Affinity Gel (651502) antibodies were purchased from BioLegend. Secondary Alexa Fluor–labeled antibodies were from Jackson ImmunoResearch. Protein G (L00209) resin was from GenScript. Recombinant mouse EphB4 (extracellular domain residue 16-539)-Fc protein (446-B4-200) was purchased from R&D Systems, Inc.

Li W., Wen L., Rathod B., Gingras A.C., Ley K, & Lee H.S. (2022). Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development. Life Science Alliance, 6(3), e202201800.

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Visualizing Cytoskeletal Proteins in Cells

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Phalloidin-Alexa Fluor 647 was from Invitrogen (Zwijndrecht, The Netherlands), mouse anti-Clathrin heavy chain antibody (X22) was from Thermo Scientific (Breda, The Netherlands), mouse anti-mDia1 and mouse anti-paxillin were from BD Transduction Laboratories (Breda, The Netherlands), mouse anti-mDia1 (D3) was from Santa Cruz (Heidelberg, Germany), mouse anti-tubulin was from Sigma (Zwijndrecht, The Netherlands), rabbit anti-vimentin was from GeneTex (Irvine, CA, USA), mouse anti-WAVE2 antibody was generated in-house (Isogai et al., 2015 (link)).

Leyton-Puig D., Kedziora K.M., Isogai T., van den Broek B., Jalink K, & Innocenti M. (2016). PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins. Biology Open, 5(7), 1001-1009.

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Antibodies and Dilutions for Immunofluorescence and Western Blotting

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The sources of antibodies and the dilutions used in experiments were as follows: rabbit anti-β-PIX (Millipore 07-1450, Darmstadt, Germany; dilution for Western blotting: 1/1000; dilution for immunofluorescence: 1/500); mouse anti-paxillin (BD Bioscience 610052, San Jose, California, USA; dilution for immunofluorescence: 1/1000); mouse anti-GAPDH (GeneTex GTX627408, Hsinchu, Taiwan; dilution for Western blotting: 1/1000); DNase I (Sigma-Aldrich SAB2702031, Saint Louis, Missouri, USA; dilution for immunofluorescence: 1:1000); Alexa Fuor 488-antimouse IgG (Jackson ImmunoResearch 715-546-151/J1, West Grove, Philadelphia, USA; dilution for immunofluorescence: 1/300); Alexa Fuor 568-antimouse IgG (Invitrogen A11031; dilution for immunofluorescence: 1/300); Alexa Fuor 568-anti-rabbit IgG (Invitrogen A11036; dilution for immunofluorescence: 1/300); Alexa Fuor 647-antimouse IgG (Jackson ImmunoResearch 715-606-151/J1; dilution for immunofluorescence: 1/300). Alexa Fluor 488 phalloidin (A12379; dilution for immunofluorescence: 1/400) and Alexa Fluor 568 phalloidin (A12380; dilution for immunofluorescence: 1/400) were obtained from Invitrogen. HRP-AffiniPure goat antimouse IgG (115-035-174; dilution for Western blotting: 1/5000) and HRP-AffiniPure goat anti-rabbit IgG (211-032-171; dilution for Western blotting: 1/5000) were obtained from Jackson ImmunoResearch.

Yu H.W., Chen Y.Q., Huang C.M., Liu C.Y., Chiou A., Wang Y.K., Tang M.J, & Kuo J.C. (2015). β-PIX controls intracellular viscoelasticity to regulate lung cancer cell migration. Journal of Cellular and Molecular Medicine, 19(5), 934-947.

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Cell Migration Assay Reagents

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Rabbit anti–β1 integrin, goat anti-CAPN1, and rabbit anti-FAK were from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-ezrin was purchased from Cell Signaling (Beverly, MA). Rabbit anti–phospho-Y397 FAK and mouse anti-paxillin were from BD Transduction Labs (Mississauga, Canada). Mouse anti-cortactin was purchased from EMD Millipore (Etobicoke, Canada). Mouse anti–γ-tubulin, mouse anti-moesin, mouse anti-vinculin, mouse anti-talin, and rabbit anti-zyxin were purchased from Sigma-Aldrich (Oakville, Canada). Rabbit anti–calpain-2, which recognizes both CAPN2 and CAPNS1, was provided by P. Greer (Queen’s University, Kingston, Canada; Arthur et al., 2000 (link)). Transwell chambers (8-μm pore size) were obtained from Corning (Mississauga, Canada). Eight-well chamber slides were purchased from LabTek (Mississauga, Canada). μ-Slide VI, 35-mm μ-Dish, and eight-well μ-Slides were from Ibidi (Madison, WI). Alexa Fluor reagents and cell trackers were obtained from Life Technologies (Burlington, Canada). Growth factor–reduced Matrigel and rat-tail collagen I were purchased from BD Biosciences (Mississauga, Canada). Fibronectin, gelatin, and puromycin were from Sigma-Aldrich.

Hoskin V., Szeto A., Ghaffari A., Greer P.A., Côté G.P, & Elliott B.E. (2015). Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion. Molecular Biology of the Cell, 26(19), 3464-3479.

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Antibody-based Protein Detection Assay

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Rabbit anti-PRPF4B (8577, Cell Signaling Technology), mouse anti-Tubulin (T-9026, Sigma–Aldrich), mouse anti-Paxillin (610052, BD Biosciences), mouse anti-ITGB1 (610467, BD Biosciences), mouse anti-FAK (610087, BD Biosciences), mouse anti-N-cadherin (610920, BD Biosciences), mouse anti-E-cadherin (610181, BD Biosciences), rabbit anti-p-FAKY397 (446–24ZG, Thermo Fisher), and mouse anti-Vimentin (ab8069, Abcam) were all commercially purchased. All antibodies were used in a 1:1,000 dilution. Rabbit anti-ITGA3 and rabbit anti-Laminin5 were kindly provided by A. Sonnenberg (NKI, Amsterdam, The Netherlands). Anti-mouse and anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Jackson ImmunoResearch.

Koedoot E., Fokkelman M., Rogkoti V.M., Smid M., van de Sandt I., de Bont H., Pont C., Klip J.E., Wink S., Timmermans M.A., Wiemer E.A., Stoilov P., Foekens J.A., Le Dévédec S.E., Martens J.W, & van de Water B. (2019). Uncovering the signaling landscape controlling breast cancer cell migration identifies novel metastasis driver genes. Nature Communications, 10, 2983.

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Immunofluorescence Staining of Focal Adhesions

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FAK inhibitor PF-228 (PZ0117) was purchased from Sigma. Alexa Fluor 488-phalloidin (A12379), Alexa Fluor 568-phalloidin (A12380), Alexa Fluor 488-goat anti-mouse (A11029), Alexa Fluor 488-goat anti-rabbit (A11034), Alexa Fluor 568-goat-anti-rabbit (A11011), and Alexa Fluor 568-goat anti-mouse (A11004) antibodies were purchased from Life Technologies (Grand Island, NY).
Mouse anti-Paxillin (1:200, 610051) and mouse anti-FAK (1:500, 610088) antibodies were purchased from BD Biosciences. Rabbit anti-FAK pY357 (1:200, ab81298) was purchased from Abcam. Mouse anti-α-actinin 2 (1:200, A7811) was purchased from Sigma Aldrich. Titin (9D10) antibody was purchased from the Developmental Studies Hybridoma Bank (University of Iowa). Primary antibody conjugation was performed using Mix-n-Stain kits (92233) purchased from Biotium (Fremont, CA) according to the instructions provided by the manufacturer. Paraformaldehyde (PFA, 15710) was purchased from Electron Microscopy Sciences (Hatfield, PA). Triton X-100 (BP151100) was purchased from Fisher Scientific (Suwanee, GA).

Taneja N., Neininger A.C, & Burnette D.T. (2020). Coupling to substrate adhesions drives the maturation of muscle stress fibers into myofibrils within cardiomyocytes. Molecular Biology of the Cell, 31(12), 1273-1288.

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Immunofluorescence and Immunoprecipitation Protocols

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Cells were fixed in methanol/acetone (1:1) with exception of samples processed for Rac-GTP labeling, which were fixed in 2% formalin and permeabilized with 0.1% Triton X-100. Processing of samples was carried out as described previously.^{19 (link)} Antibodies used for immunofluorescence were: rabbit anti-GIT2^{19 (link)}; rabbit anti-DOCK5 (described above); rabbit anti-DOCK1 (Santa Cruz Biotechnology, H-70); mouse anti-paxillin (BD Biosciences, cl. 349), mouse anti-ARP3 (Sigma, cl. FMS338); mouse anti-Rac-GTP (NewEast Biosciences, 26903); and mouse anti-Myc epitope (Cell Signaling Technologies, clone 9B11). Confocal images were acquired using a spinning disk confocal microscope.
Antibodies used for immunoprecipitation and western blotting were: mouse anti-Crk (BD Biosciences; clone 22); rabbit anti-Crk (Santa Cruz Biotechnology; C-20), mouse anti-DOCK1 (Santa Cruz Biotechnology; clone H-4), mouse anti-p130Cas (BD Biosciences; clone 21), mouse anti-Rac1 (BD Biosciences; clone 102), rabbit anti-Rac3 (Abcam; clone EPR6680), rabbit anti-FAK (Santa Cruz Biotechnology; clone A-17), rabbit anti-phospho-p130Cas(Tyr410), rabbit anti-phospho-paxillin(Tyr118) and rabbit anti-phospho-FAK(Tyr397). All phospho-specific antibodies were from Cell Signaling Technologies.

Frank S.R., Köllmann C.P., van Lidth de Jeude J.F., Thiagarajah J.R., Engelholm L.H., Frödin M, & Hansen S.H. (2016). The focal adhesion-associated proteins DOCK5 and GIT2 comprise a rheostat in control of epithelial invasion. Oncogene, 36(13), 1816-1828.

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Immunofluorescence and Immunoprecipitation Assays

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Cells were fixed in methanol/acetone (1:1) with exception of samples processed for Rac-GTP labeling, which were fixed in 2% formalin and permeabilized with 0.1% Triton X-100. Processing of samples was carried out as described previously.^{19 (link)} Antibodies used for immunofluorescence were: rabbit anti-GIT2;^{19 (link)} rabbit anti-DOCK5 (described above); rabbit anti-DOCK1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, clone H-70); mouse anti-paxillin (BD Biosciences, San Jose, CA, USA, clone 349), mouse anti-ARP3 (Sigma, St Louis, MO, USA, clone FMS338); mouse anti-Rac-GTP (NewEast Biosciences, Malvern, PA, USA, clone 26903); and mouse anti-Myc epitope (Cell Signaling Technologies, Danvers, MA, USA, clone 9B11). Confocal images were acquired using a spinning disk confocal microscope.
Antibodies used for immunoprecipitation and western blotting were: mouse anti-Crk (BD Biosciences; clone 22); rabbit anti-Crk (Santa Cruz Biotechnology; C-20), mouse anti-DOCK1 (Santa Cruz Biotechnology; clone H-4), mouse anti-p130Cas (BD Biosciences; clone 21), mouse anti-Rac1 (BD Biosciences; clone 102), rabbit anti-Rac3 (Abcam; clone EPR6680), rabbit anti-FAK (Santa Cruz Biotechnology; clone A-17), rabbit anti-phospho-p130Cas(Tyr410), rabbit anti-phospho-paxillin(Tyr118) and rabbit anti-phospho-FAK(Tyr397). All phospho-specific antibodies were from Cell Signaling Technologies.

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Paxillin Immunofluorescence Staining

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Mouse anti-paxillin (no. 610052) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Rabbit anti-phosphorylated (pY118) paxillin (cat. number 44-722G) was from Invitrogen (ThermoFisher Scientific). Mouse anti-vinculin (V-9131) and mouse anti-tubulin (T-9026) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-GAPDH (sc-32233) was purchased from Santa Cruz (Dallas, TX, USA). Rhodamin phalloidin and goat anti-mouse Alexa-488 conjugated secondary antibodies were from Molecular Probes (Invitrogen/ThermoFisher Scientific). Goat anti-rabbit CY-3 and goat anti-mouse Alexa647 conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA, USA). Nocodazole was purchased from Fluka (Zwijndrecht, The Netherlands).

Fokkelman M., Balcıoğlu H.E., Klip J.E., Yan K., Verbeek F.J., Danen E.H, & van de Water B. (2016). Cellular adhesome screen identifies critical modulators of focal adhesion dynamics, cellular traction forces and cell migration behaviour. Scientific Reports, 6, 31707.

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Integrin-Mediated Cellular Adhesion Analysis

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Mouse anti-paxillin (BD 610052); rat anti-active integrin β1 (9EG7; BD 553715); rabbit-anti-Tom20 (Santa Cruz sc-11415); mouse-anti-MYH1/2 (Santa Cruz sc-53088); rabbit anti-fibronectin (Santa Cruz sc-9068); rabbit anti-GAPDH (GeneTex GTX100118); rabbit anti-β-actin (GeneTex GTX100313); rabbit anti-integrin β1 (GeneTex GTX128839); rabbit anti-paxillin (GeneTex GTX125891); rabbit anti-pY397-FAK (GeneTex GTX129840); rabbit anti-kinesin-1 (Abcam AB167429); rabbit anti-GFP (Abcam AB290); mouse anti-α-tubulin (Sigma T5168); mouse anti-MYH2 (Thermo Fisher 14-6503-80); rabbit anti-FAK (Thermo Fisher AHO0502); Alexa Fluor 488 phalloidin (Thermo Fisher A12379); Alexa Fluor 568 phalloidin (Thermo Fisher A12380); Alexa Fluor 488-anti-rabbit IgG (Thermo Fisher A11034); Alexa Fluor 488-anti-mouse IgG (Thermo Fisher A11029); Alexa Fluor 488-anti-rat IgG (Thermo Fisher A11006); Alexa Fluor 568-anti-rabbit IgG (Thermo Fisher A11036); Alexa Fluor 568-anti-mouse IgG (Thermo Fisher A11031); DAPI (Thermo Fisher D1306); Mitotracker Red (Thermo Fisher M7512); HRP-AffiniPure mouse anti-rabbit IgG (Jackson ImmunoResearch 211-032-171); and HRP-AffiniPure goat anti-mouse IgG (Jackson ImmunoResearch 115-035-174).

Lin J.W., Huang Y.M., Chen Y.Q., Chuang T.Y., Lan T.Y., Liu Y.W., Pan H.W., You L.R., Wang Y.K., Lin K.H., Chiou A, & Kuo J.C. (2021). Dexamethasone accelerates muscle regeneration by modulating kinesin-1-mediated focal adhesion signals. Cell Death Discovery, 7, 35.

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