Mouse anti paxillin antibody

Manufactured by BD

Sourced in United States

The mouse anti-paxillin antibody is a laboratory reagent used to detect the paxillin protein in various cell and tissue samples. Paxillin is an adapter protein involved in the regulation of cell adhesion and cytoskeleton organization. This antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunocytochemistry to study the expression and localization of paxillin in biological systems.

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8 protocols using mouse anti paxillin antibody

E-cadherin Perturbation and Focal Adhesion Visualization

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In experiments where antibodies were used to perturb E-cadherin-mediated cell–cell adhesions, MCF10A-E-cadherin-GFP cells were treated with 3 µg/ml of neutral, control antibody (76D5; gift of BM Gumbiner (Petrova et al., 2012 (link))), or E-cadherin-blocking antibody (DECMA-1; Abcam) for at least 2 hr prior to TFM measurements. As an additional control, cells were also imaged and measured pre-treatment.
In immunostaining experiments to visualize focal adhesions in MCF10A-E-cadherin-GFP cell clusters, a purified mouse anti-paxillin antibody (BD Biosciences, San Jose, CA) was used as the primary antibody and the Alexa Fluor 568 goat anti-mouse antibody (Life Technologies, Grand Island, NY) was used as the secondary antibody.

Ng M.R., Besser A., Brugge J.S, & Danuser G. (2014). Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters. eLife, 3, e03282.

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Immunofluorescence Analysis of Cell Adhesion

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The mouse anti-paxillin antibody was purchased from BD Biosciences (610052) and the corresponding secondary Alexa488 anti-mouse antibody from ThermoFisher Scientific (A-11029). The rabbit anti-fibronectin antibody was obtained from Sigma (F3648) and the corresponding secondary Alexa647 anti-rabbit antibody from ThermoFisher Scientific (A-21245). Alexa Fluor 568-coupled phalloidin was from ThermoFisher Scientific (A12380). Rat anti-E-cadherin antibody was obtained from ThermoFisher Scientific (13-1900), rabbit anti-ZO1 antibody from ThermoFisher Scientific (61-7300), mouse anti-vimentin antibody from Sigma (V2258), mouse anti-SMAD2/3 from BD Biosciences (610842), rabbit anti-p-SMAD3 from Cell Signaling (p-Ser423/425; 9520) and mouse anti-GAPDH from Sigma (G8795). For quantitative immunoblot analysis, secondary antibodies from LI-COR Biosciences, IRDye 680RD Goat anti-Mouse (926-68070) and IRDye 800CW Goat anti-Rabbit (926-32211) were used. DAPI was acquired from Sigma (D9542), recombinant human TGFβ1 protein from R & D Systems (240B-0-10), 16% paraformaldehyd (PFA) from Electron Microscopy Services (15710-S), fatty-acid free BSA from Calbiochem (126575), Trypsin/EDTA from Sigma (T4174), PBS from Gibco (14200-067), and Triton X-100 from Sigma (X-100).

Lotz-Jenne C., Lüthi U., Ackerknecht S., Lehembre F., Fink T., Stritt M., Wirth M., Pavan S., Bill R., Regenass U., Christofori G, & Meyer-Schaller N. (2016). A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor. Oncotarget, 7(18), 25983-26002.

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Purification of Recombinant Protein Tyrosine Phosphatases

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Recombinant proteins corresponding to the entire intracellular regions (ICRs) of PTPRZ1, PTPRA, and PTPRM were expressed using a baculovirus-silkworm expression system, and purified as described^{6 (link)}. The ICRs of PTPRG, PTPRS, and PTPRB and the catalytic domains of PTPN1 and PTPN6 were expressed as glutathione-S-transferase (GST) fusion proteins from each pGEX plasmid in Escherichia coli strain BL21 (ref. 6 (link)). GST fusion proteins were purified by glutathione affinity chromatography as described^{43 (link)}. Chondroitinase ABC (chABC) was purchased from Sigma-Aldrich (catalog #C3667). Anti-PTPRZ-S, rabbit polyclonal antibodies against the extracellular region of PTPRZ was described previously (ref. 44 (link)). The following are the specificities and sources of the commercially available antibodies used in the present study: Anti-RPTPβ (a monoclonal antibody against the intracellular domain of PTPRZ1 receptors, #610179, BD Biosciences), anti-SOX2 (#ab97959, Abcam), anti-POU3F2 (#12137, Cell Signaling), anti-OLIG2 (#AB9610, Millipore), anti-SALL2 (#12679–1-AP, Proteintech Group), anti-GAPDH (#ab9482, Abcam) anti-phosphotyrosine (PY20; #ab16389, Abcam), and anti-pY118-paxillin (#2541, Cell Signaling), mouse anti-paxillin antibody (#610569, BD Bioscience), anti-MBP (#sc-13914, Santa Cruz Biotechnology), and anti-NG2 proteoglycan (#AB5320, Millipore).

Fujikawa A., Sugawara H., Tanaka T., Matsumoto M., Kuboyama K., Suzuki R., Tanga N., Ogata A., Masumura M, & Noda M. (2017). Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells. Scientific Reports, 7, 5609.

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Proximal Protein Crosslinking and Immunoprecipitation

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For immunoprecipitations, cells were washed twice with PBS, and 0.5 mM dithiobis(succinimidyl propionate) (Thermo Fisher Scientific) dissolved in PBS was added for 30 min at RT to cross-link proximal proteins. The reaction was stopped by incubation with 50 mM Tris, pH 7.5, for 10 min. Cells were washed with cold PBS and lysed in mammalian protein extraction reagent (Thermo Fisher Scientific) supplemented with protease inhibitors (Roche Diagnostics) and phosphatase inhibitor cocktails (Sigma-Aldrich). µMACS GFP and DYKDDDDK (to recognize the Flag-tag) Isolation kits (Miltenyi) were used according to the manufacturer’s protocol using 1–2 mg protein lysate. Endogenous paxillin immunoprecipitation was performed using 5 µg mouse anti-paxillin antibody (BD Biosciences) and Pierce Protein A/G Magnetic Beads following the user’s guide. Mouse IgG1 (Sigma-Aldrich) was used as control. Immunoprecipitations and 40 µg per loading control were subjected to 10% SDS-PAGE and subsequent Western blotting.

Klapproth S., Bromberger T., Türk C., Krüger M, & Moser M. (2019). A kindlin-3–leupaxin–paxillin signaling pathway regulates podosome stability. The Journal of Cell Biology, 218(10), 3436-3454.

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Comprehensive Protein Signaling Analysis

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Rabbit antibodies against merlin (D1D8), c-ABL, phospho-AKT (Thr308; C31E5E), cyclin-D1 (92G2), FYN, p-MEK1/2 (Ser217/221), PDGFRα and β, SRC (36D10), p-SRC family (Tyr416), p-ERK1/2 (D13.14.4E), YES and mouse antibodies recognizing AKT (40D4), β-Actin (8H10D10), MEK1/2 (L38C12), ERK1/2, p27^Kip1 (SX53G8.5) were purchased from Cell Signaling. Rabbit antibodies against p-paxillin (Tyr118) and p-FAK (Tyr577/Tyr576) were purchased from Invitrogen. Rabbit p70S6 kinase (Thr229) was purchased form ThermoFisher Scientific. Rabbit anti-p-ABL (Tyr245), and human nuclear antigen antibodies were obtained from Millipore and antibodies for myelin- proteolipid protein and GAP43 were from Abcam. The anti-S100 antibody was purchased from Dako. Mouse anti-paxillin antibody was from BD bioscience. Secondary antibodies, goat anti-rabbit IgG conjugated with DyLight 800 4X-PEG, goat anti-Mouse IgG conjugated with DyLight 680, were purchased from Cell Signaling. Ponatinib, perifosine/KRX-0401, and selumetinib/AZD6244 were purchased from SelleckChem. The STAT3 inhibitor S3I-201/NSC-74859 was purchased from MedChem Express.

Petrilli A.M., Garcia J., Bott M., Plati S.K., Dinh C.T., Bracho O.R., Yan D., Zou B., Mittal R., Telischi F.F., Liu X.Z., Chang L.S., Bradley D.W., Copik A.J, & Fernández-Valle C. (2017). Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells. Oncotarget, 8(19), 31666-31681.

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Cyclic RGDfk-NH2 Cell Adhesion Assay

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Cyclic peptide RGDfk-NH2 was obtained from Peptides International (Louisvile, KY, USA), biotin-labeled bovine serum albumin (BSA), Hoechst-33342, and glutaraldehyde were from Sigma-Aldrich (Louis, MO, USA), mouse anti-paxillin antibody and fibronectin were from BD Biosciences (San Jose, CA, USA), Neutravidin and Sulfo-SMCC were from Thermo Fisher Scientific (Waltham, MA, USA), Phalloidin was from molecular probe, (Grand Island, NY, USA), mouse anti-YAP monoclonal IgG was from Santa Cruz Biotechnology (Dallas, Texas, TX, USA), CF568 goat anti-mouse IgG was from Biotium, Inc. (Fremont, CA, USA).

Chang Chien C.Y., Chou S.H, & Lee H.H. (2022). Integrin molecular tension required for focal adhesion maturation and YAP nuclear translocation. Biochemistry and Biophysics Reports, 31, 101287.

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Immunohistochemistry and Proximity Ligation Assay

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For immunohistochemistry experiments, explants were fixed with 4% PFA for 10 mins at RT, followed by permeabilization with 0.1% NP40 for 30 mins at 37°C. Next, the cells were blocked with 1% BSA in PBS for 30 mins at 37°C, prior to incubation with the primary antibody for 1h at 37°C. Primary antibodies were prepared in blocking solution, and were used in the following dilutions: mouse anti-E-cadherin antibody (BD Biosciences) (1:200), mouse anti-Paxillin antibody (BD Biosciences) (1:200), and rabbit anti-Active Yap1 (Abcam)(1:200), anti-pH3(S10) (Abcam, 1:200), anti-Caspase3 (R&D Systems, 1:200). Following primary antibody incubation, the explants were washed 4X times in PBS for 10 mins and incubated in a secondary antibody cocktail for 30 mins at 37°C. Finally, cells were washed 3X times in PBS and stained with DAPI or Phalloidin for 20 mins at RT. Post antibody staining, imaging was performed using Andor/Olympus Spinning Disk Confocal microscope at BRC facility, Cornell University.For Proximity Ligation Assay (PLA), explants were fixed and permeabilized as described above.The primary antibodies used were: Mouse Anti-YAP1 (DSHB) (1:5) and Rabbit Anti-TEAD1 (Abcam) (1:200). Following primary antibody incubation, the remaining steps of PLA were performed using reagents from the Duolink PLA detection kit (Sigma Aldrich, DUO92101) according to the manufacturer’s protocol.

Bhattacharya D., Azambuja A.P, & Simoes-Costa M. (2020). Metabolic reprogramming promotes neural crest migration via Yap/Tead signaling. Developmental cell, 53(2), 199-211.e6.

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Co-Immunoprecipitation of Cytoskeletal Proteins

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Cells at a confluence of 70% were lysed with 1 mL ice-cold lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris HCl, pH 8.0) supplemented with protease and phosphatase inhibitors. Following a 30-min incubation period, cells were centrifuged (10,000× g for 10 min at 4 °C) and the supernatant collected. Co-IP experiments were performed as described before [13 (link)]. In brief, to pull down CB, 2–4 µg of rabbit anti-CB (Swant) was used, then 100 µL of µMACS protein A MicroBeads (Miltenyi Biotec, Auburn, AL, USA) were added to the lysate and incubated at 4 °C for 30 min. Samples were loaded on MACS separation columns (Miltenyi Biotec) and subjected to magnetic immunoprecipitation. The columns were washed 3 times with a wash buffer and protein complexes were eluted in 50 µL of pre-warmed SDS gel loading buffer 1X, subjected to electrophoresis (SDS-PAGE) and subsequent Western blotting. Membranes were probed with the antibodies rabbit anti-CB (1:10,000, Swant), rabbit anti-FAK (1:1000, Cell Signaling Technology), and rabbit anti-septin 7 (Bethyl Laboratories Inc., Montgomery, TX, USA); a mouse anti-paxillin antibody (1:2000; BD Bioscience, Allschwil, Switzerland) served as a negative control.

Wörthmüller J., Oberson A., Salicio V., Blum W, & Schwaller B. (2018). Calretinin Functions in Malignant Mesothelioma Cells Cannot Be Replaced by the Closely Related Ca2+-Binding Proteins Calbindin-D28k and Parvalbumin. International Journal of Molecular Sciences, 19(12), 4015.

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