Human pulmonary artery endothelial cells, small airway epithelial cells (SAEC), and cell culture basal medium with growth supplements were obtained from Lonza (Allendale, NJ). Cells were cultured according to the manufacturer’s protocol and used at passages 5–7. All reagents for immunofluorescence studies were purchased from Molecular Probes (Eugene, OR). PGA2, EP4 agonist CAY10580, and prostaglandin receptor inhibitors were obtained from Cayman (Ann Arbor, MI). The following receptor inhibitors were used: TP inhibitor SQ29548, DP inhibitor BWA868C, IP inhibitor CAY10449, EP4 inhibitors L161982 and GW627368X, FP inhibitor AL8810, and EP1-3 inhibitor AH6809. Rac1 inhibitor NAS23766 was obtained from EMD Millipore (Billerica, MA); PKA peptide inhibitor PKI was from Promega (Madison, WI). VE-cadherin antibody was obtained from Cayman; EP4, Rap1, Rac1, RhoA, ICAM1, and VCAM1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); β-actin and β-tubulin antibodies were from Sigma-Aldrich (St. Louis, MO); cortactin, phospho-cortactin, VASP, phospho-VASP, phospho-CREB, phospho-MYPT, diphospho-MLC, phospho-NFκB, and IκBα antibodies were obtained from Cell Signaling (Beverly, MA); α-catenin, p120-catenin, and ZO-1 antibodies were from BD Transduction Laboratories (San Diego, CA). Unless specified, other biochemical reagents were obtained from Sigma-Aldrich.
P120 catenin
Manufactured by BD
Sourced in United States
P120-catenin is a laboratory equipment product. It is a protein that plays a role in cell-cell adhesion and signaling. The core function of P120-catenin is to regulate the stability and function of cadherin cell adhesion proteins.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by BD (6 mentions)
β catenin, by BD (5 mentions)
α catenin, by BD (2 mentions)
N cadherin, by BD (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
α catenin, by Merck Group (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
P thr 423 pak1, by Cell Signaling Technology (2 mentions)
Crk 2, by Santa Cruz Biotechnology (2 mentions)
P ser41 crk 2, by Santa Cruz Biotechnology (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Phospho nf κb, by Cell Signaling Technology (1 mentions)
Phospho mypt, by Cell Signaling Technology (1 mentions)
Phospho creb, by Cell Signaling Technology (1 mentions)
Cortactin, by Cell Signaling Technology (1 mentions)
Phospho cortactin, by Cell Signaling Technology (1 mentions)
Ve cadherin antibody, by Cayman Chemical (1 mentions)
Phospho vasp, by Cell Signaling Technology (1 mentions)
Vcam 1, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Merck Group (1 mentions)
15 protocols using p120 catenin
1
Endothelial and Epithelial Cell Culture
2
Western Blotting and Immunoprecipitation Assays
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Western blotting and immunoprecipitation assays were performed as described previously [33 (link)]. Primary antibodies against THUMPD1 and LaminB1 (1:100) were purchased from Santa Cruz Biotechnology, Inc., and antibodies against GAPDH (1:500 and 1:5,000) were from Sigma (St. Louis, MO, USA). PI3 kinase inhibitor LY294002 (10 μM) and antibodies against Snail, Slug, YAP, p-YAP-Ser127, LATS, p-LATS (Ser1079), Myc-tag, Vimentin, P38, p-P38, ERK, p-ERK, JNK, p-JNK, GSK3β, p-GSK3β (Ser9), p-P65 (Ser536), AKT, p-AKT, and active β-catenin (1:1,000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-catenin, E-cadherin, N-cadherin, P65, and P120 catenin (1:1,000) were purchased from BD Transduction Laboratories (Lexington, KY, USA). Antibodies against Zo-1 and Occludin (1:500) were purchased from Proteintech (Chicago, IL, USA). Proteasome inhibitor MG132 (10μM) and α-tublin antibody (1:500) were purchased from Beyotime (Jiangsu, China).
3
Antibody Detection and Characterization
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Primary antibodies were used that recognise N-cadherin (BD transduction); α-catenin (Sigma C2081); β-catenin (BD transductions 610920); p120-catenin (BD Transduction 61034); ERK1/2 (Sigma M5670), mCherry (Abcam ab183628; western blotting); mCherry (Life Technologies M11217; Immunoprecipitation), AKT 1/2/3 (Santa Cruz), Slit2 (Abcam ab134166; western blotting), Slit2 (Thermo Fisher Scientific PA531133; immunofluorescence), Slit3 (Sigma SAB2104337; immunofluorescence) Slit3 (R&D Systems AF3629; western blotting), Myc (Merck Millipore 05-724). Alexa Fluor secondary antibodies were obtained from Invitrogen. Horseradish peroxidase (HRP)-linked antibodies were obtained from GE Healthcare.
4
Western Blot Analysis of Cell Junctions
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Cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor tablets (Roche, IN, USA). Cell lysates were cleared by centrifugation and protein concentration determined by Bio-Rad Protein Assay kit (Bio-Rad Laboratories, CA, USA). 5–30 µg (as indicated in the figure legends) of total protein in SDS sample buffer was loaded per lane and separated on NuPAGE Tris-Acetate precast polyacrylamide gels (Life Technologies). Detection of proteins was done with the following primary Abs and dilutions: β-catenin CTD (BD Transduction Laboratories, #610153, 1∶2000), γ-catenin (BD Transduction Laboratories, #610253, 1∶5000) β-catenin NTD (Abcam, ab32572, 1∶5000), E-cadherin (BD Transduction Laboratories, #610181, 1∶4000), α-Catenin (BD Transduction Laboratories, #610193, 1∶500), p120-catenin (BD Transduction Laboratories, #610133, 1∶2000), Actin (Sigma-Aldrich, A2066, 1∶2000). Secondary Abs were: donkey anti-mouse IgG-HRP (Santa Cruz Biotechnology, CA, USA, sc-2314, 1∶5000), donkey anti-rabbit IgG-HRP (Santa Cruz biotechnology, sc-2313, 1∶5000). Signals were developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and quantification of signal intensities was done using the Image Studio Lite software (LI-COR, NB, USA).
5
Immunohistochemical Analysis of Retinal Cell Markers
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The following primary antibodies were used: glutamine synthetase (1:250; BD Biosciences), rhodopsin (1:500; Millipore), cone arrestin (1:500; Millipore), PKCα (1:250; BD Biosciences), MPP4 AK4 (1:200; homemade34 (link)), CRB1 AK2 (pH 1.5) (1:200; homemade8 (link)), CRB2 (1:2008 (link)), p120-catenin (1:100; BD Biosciences), GFAP (1:200; Dako), CD11b (1:100; BD Biosciences); PLVAP (1:200; BD Pharmingen), and VE-cadherin (1:100; BD Biosciences).
6
Frizzled-7 Knockdown Modulates Cytoskeletal Dynamics
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The cells were grown on glass coverslips in 6-well plates and then transfected with one of two FZD7 siRNA sequences (FZD7-5 and FZD7-6) or the non-targeting negative control siRNA for 48 h. For staining of F-actin, β-catenin, p120 catenin and pMLC (Ser19), the transfected cells were washed two times with PBS to remove residual media/serum, fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilised with 0.05% Triton X-100 for 5 min at room temperature. For CK1ɛ, DVL2 pSer143 and pan-cadherin staining, the cells were washed two times with PBS and fixed with ice-cold methanol for 10 min at −20 °C. Primary antibodies were used at the following concentrations: 1 : 100 for β-catenin (BD Biosciences), p120 catenin (BD Biosciences), DVL2 pS143 (GeneTex, Irvine, CA, USA), CK1ɛ (Santa Cruz Biotechnology Inc., Dallas, TX, USA), pan-cadherin (Abcam, Cambridge, MA, USA) and pMLC2 (Ser19) (Cell Signaling Technology, Beverly, MA, USA); 1 : 200 for rhodamine phalloidin F-actin (Sigma-Aldrich); and 1 : 1000 for γ-tubulin (Sigma-Aldrich). Alexa Fluor-488 and -594 secondary anti-mouse and anti-rabbit antibodies (Invitrogen, Eugene, OR, USA) were used at 1 : 1000 dilutions. Cells were mounted with Vectashield mounting medium containing DAPI (Vector Laboratories Inc., Burlingame, CA, USA).
7
SDS-PAGE Analysis of Cellular Proteins
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Protein lysates were prepared for SDS–PAGE analysis by washing cells twice with PBS and then adding sample buffer with urea (2% SDS, 10% glycerol, 0.001% bromophenolblue, 0.1 M DTT, 0.0625 M Tris/pH 6.8 and 6 M urea). After homogenizing three times with a 23-gauge microneedle, samples were heated at 100 °C for 10 min. The following primary antibodies were used to detect proteins: α-tubulin (mouse, 1:20) (ref. 66 (link)), E-cadherin (BD Biosciences—610182, mouse, 1:1,000); p120-catenin (BD Biosciences—610134, mouse, 1:1,000); Kif1B (Bethyl Laboratories—A301-056A, Inc., rabbit, 1:1,000); DLC2 (Santa Cruz—sc67843, goat, 1:200); mDia3 (ECM Bioscience—DP4511, rabbit, 1:1,000); phospho-Aurora B (T232) (Abcam—ab61074, rabbit, 1:200); Cdc42 (Santa Cruz—sc89, mouse, 1:100). Uncropped versions of all blots are found in Supplementary Fig. 11 .
8
Protein Interactions via Co-Immunoprecipitation
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Co-Immunoprecipitations (Co-IP) were performed using Dynabeads (Life Technologies) according to the manufacturer’s protocol. β-Catenin (BD Biosciences #610153, dilution 1:1000) or Lasp1 antibody (Abcam #ab130109, dilution 1:1000) was coupled to protein G–labelled Dynabeads. Equal amounts of cell lysates were transferred to the antibody-bead complex and incubated overnight at 4 °C. Antibodies against the following proteins were detected by immunoblotting: β-Actin (Abcam #ab8227, dilution 1:1000), β-Catenin (Cell Signaling #9562, dilution 1:1000), Cadherin-11 (Life Technologies #321700, dilution 1:1000), Lasp1 (Abcam #ab130109, dilution 1:2000), p120-Catenin (BD Biosciences #610134, dilution 1:1000) and GAPDH (Cell Signaling #3638 S, dilution 1:1000).
9
Construct Generation and Antibody Usage
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The generation of the 90K-myc and ISG15-V5 constructs was described previously [19 (link)]. HA-Ub was kindly provided by K.Y. Lee (Chonnam National University, Gwangju, Korea). The antibodies used in the present study were as follows: anti-E-cadherin (C20820, BD Biosciences, San Jose, CA, USA; CS#3195, Cell Signaling Technology, Danvers, MA, USA), p120-catenin (610133, BD Biosciences), β-catenin (#9582, Cell Signaling Technology), actin (A2066, Sigma, St. Louis, MO, USA), myc (M047-3, MBL, Woburn, MA, USA), V5 (PM003, MBL), HA (H9658, Sigma), tubulin (sc-5286, Santa Cruz Biotech, Dallas, TX, USA), phospho-serine (P3430, Sigma), phospho-tyrosine (05-321, Upstate, Molsheim, France), phospho-threonine (P3555, Sigma), phospho-Tyr-228–p120-catenin (ab32403, abcam, Cambridge, MA, USA).
10
Antibodies Used in Wnt Signaling Study
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