P120 catenin

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P120-catenin is a cytoplasmic protein that plays a role in cell-cell adhesion. It binds to the cytoplasmic domain of cadherin proteins and is involved in the regulation of cadherin turnover and actin cytoskeleton organization.

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Anti-p120-catenin (2 citations) Rabbit anti-phospho-p120-Catenin (2 citations)

7 protocols using p120 catenin

Pulmonary Endothelial Cell Culture Protocol

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Human pulmonary artery endothelial cells (HPAECs) were obtained from Lonza (Allendale, NJ) and used at passages 5–8. All experiments were performed in EGM growth medium (Lonza) containing 2% fetal bovine serum (FBS) unless otherwise specified. Texas Red–conjugated phalloidin and Alexa Fluor 488–labeled secondary antibodies were purchased form Molecular Probes (Eugene, OR). TRAP6 was obtained from AnaSpec (San Jose, CA). 8CPT was obtained from Calbiochem (La Jolla, CA). GGTI-298 and thrombin were obtained from Millipore-Sigma. Antibodies to diphospho–myosin light chain (MLC), pan-MLC, MYPT, pMYPT, p120-catenin, phospho-NFκB, β-actin, and tubulin antibodies were obtained from Cell Signaling (Beverly, MA); Rap1, VE-cadherin, ICAM1, VCAM1, and E-selectin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-linked anti-mouse and anti-rabbit IgG were obtained from Cell Signaling (Beverly, MA). Unless otherwise specified, all biochemical reagents were obtained from Sigma (St. Louis, MO).

Ke Y., Karki P., Zhang C., Li Y., Nguyen T., Birukov K.G, & Birukova A.A. (2019). Mechanosensitive Rap1 activation promotes barrier function of lung vascular endothelium under cyclic stretch. Molecular Biology of the Cell, 30(8), 959-974.

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Membrane Protein and Cellular Fractionation

Cited 2 times

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ProteoExtract Native membrane protein extraction kit (EMD Biosciences Inc., San Diego, CA) was used for enrichment of membrane proteins, whereas NE-PER nuclear and cytoplasmic reagents (Pierce Biotechnology, Rockford, IL) were used to separate cytoplasmic and nuclear fractions. The basic immunoblotting methodology was as reported [25 (link)–28 (link)], with primary antibodies to β-catenin (#9581, Cell Signaling Technology, Danvers, MA), E-cadherin (ab53226, Abcam, Cambridge, MA), p120-catenin (#4989, Cell Signaling; #339600, Zymed Laboratories Inc., San Francisco, CA), p53 (sc-126, Santa Cruz Biotechnology, Dallas, TX), β-actin (AC-74, Sigma-Aldrich, St. Louis, MO), and histone H1 (sc-8030, Santa Cruz).

Wang R., Chen Y.S., Dashwood W.M., Li Q., Löhr C.V., Fischer K., Ho E., Williams D.E, & Dashwood R.H. (2017). Divergent roles of p120-catenin isoforms linked to altered cell viability, proliferation, and invasiveness in carcinogen-induced rat skin tumors. Molecular carcinogenesis, 56(7), 1733-1742.

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Western Blot Analysis of Cell Adhesion Proteins

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Western blot analysis was performed using primary antibodies against p120 catenin (1:1000; Cell Signaling Technologies, #59854), total β-catenin (1:1000; Cell Signaling Technologies, #8480), N-cadherin (1:1000; Cell Signaling Technologies, #13116), E-cadherin (1:1000; Cell Signaling Technologies, #3195), plakoglobin (1:1000; Cell Signaling Technologies, #2309), Cyclin D2 (1:1000; Cell Signaling Technologies, #D52F9), p-ser 3 cofilin (1:1000; Cell Signaling Technologies, #3313), total-cofilin (1:1000; Cell Signaling Technologies, #5175), α-tubulin (1:20,000; Sigma–Aldrich, T6074), and β-actin (1:20,000; Sigma–Aldrich, A1978). After overnight incubation at 4 °C with aforementioned primary antibodies, membranes were washed and incubated with respective secondary antibodies anti-rabbit IgG horseradish peroxidase (1:10,000 Santa Cruz biotechnology), antimouse IgG horseradish peroxidase (1:20,000; Sigma–Aldrich) for 1 h at room temperature (RT) and developed with Clarity Western ECL substrate (Bio-Rad Laboratories).

Dissanayake W.C, & Shepherd P.R. (2022). β-Cells retain a pool of insulin-containing secretory vesicles regulated by adherens junctions and the cadherin-binding protein p120 catenin. The Journal of Biological Chemistry, 298(8), 102240.

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Detecting Epithelial Cell Junctional Protein Cleavage

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Whole-cell lysates from coinfected epithelial cells were prepared as previously described (55 ). Briefly, A549 cells were infected with IAV in the presence or absence of SB-431542, a TGF-β inhibitor. At 7 h after infection, the cells were lysed with Laemmli gel loading buffer containing 6% 2-mercaptoethanol. Cleavage of junctional proteins was detected by western blotting using specific antibodies against E-cadherin (mouse MAb; Thermo Scientific), p120-catenin (rabbit PAb; Cell Signaling), Snail1 (mouse MAb; Cell Signaling), and β-actin (rabbit PAb; Cell Signaling). Horseradish peroxidase (HRP)-conjugated antibodies against mouse or rabbit IgG (Cell Signaling) were used as the secondary antibodies.

Sumitomo T., Nakata M., Nagase S., Takahara Y., Honda-Ogawa M., Mori Y., Akamatsu Y., Yamaguchi M., Okamoto S, & Kawabata S. (2021). GP96 Drives Exacerbation of Secondary Bacterial Pneumonia following Influenza A Virus Infection. mBio, 12(3), e03269-20.

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Multicolor Immunofluorescence Imaging of Murine Colon

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For the murine studies, 8-μm frozen segments of colon from WT and ΔIEC mice were fixed in cold acetone for 15 min at −20 °C, and then 3% BSA was used for blocking at 25 °C for 1 h. Anti-E-cadherin (1:100), NDRG2 (1:80), β-catenin (1:100), P120 catenin (1:100) and ZO-1 (Cell Signaling, 8193, AB_10898025, 1:100) was used for incubation at 4 °C overnight, and Alexa-Fluor 488- or 647- conjugated antibody (Invitrogen, AB_2633280, AB_2633282, 1:2000) was used as the secondary antibody and incubated for 1 h at 25 °C. Incubation with 1 μg/ml of DAPI was performed for 15 min. Fluorescence analysis was performed with a laser scanning confocal microscope A1 (Nikon). For cellular protein distribution detection, cells were seeded in rat tail collagen-coated dishes and transfected with the indicated plasmid for 24 h, followed by gentle washing with PBS for 5 min and fixation in 4% paraformaldehyde for 30 min. Immunofluorescence staining was performed according to the above-described method. Anti-HA (1:500), anti-FLAG (1:500) and anti-SNAIL (1:200) were used for incubation at 4 °C overnight.

Wei M., Ma Y., Shen L., Xu Y., Liu L., Bu X., Guo Z., Qin H., Li Z., Wang Z., Wu K., Yao L., Li J, & Zhang J. (2020). NDRG2 regulates adherens junction integrity to restrict colitis and tumourigenesis. EBioMedicine, 61, 103068.

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Lymphangiogenesis and Lipid Metabolism Markers

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Primary antibody specific for lacteal marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1; #67538), p120-catenin (#59854), and Vascular cell adhesion molecule 1 (VCAM1; #39036) from cell signaling (Danvers, MA, USA), Zonula occludens-1 (ZO1; #sc-33725) from Santa Cruz Biotechnology (Dallas, TX, USA), Plasmalemma vesicle-associated protein 1 (PLVAP1; #NB100-77668), and ATP-binding cassette subfamily G member 1 (ABCG1; #NB400-132) from Novus (Centennial, CO) were purchased. The ELISA assay kits for LDL-cholesterol (#79980) and HDL-cholesterol (#79990) were purchased from Crystal Chem (Elk Grove Village, IL, USA). Triglyceride assay (#10010303) and Free Fatty Acid assay (#STA-618) kits were purchased from Cayman chemicals (Ann Arbor, MI, USA) and Cell Biolabs, Inc. (San Diego, CA, USA), respectively.

Prasad R., Adu-Agyeiwaah Y., Floyd J.L., Asare-Bediako B., Li Calzi S., Chakraborty D., Harbour A., Rohella A., Busik J.V., Li Q, & Grant M.B. (2023). Sustained ACE2 Expression by Probiotic Improves Integrity of Intestinal Lymphatics and Retinopathy in Type 1 Diabetic Model. Journal of Clinical Medicine, 12(5), 1771.

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Immunohistochemistry for Cell-Cell Adhesion Markers

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Immunohistochemistry (IHC) for Pax7 (Developmental Studies Hybridoma Bank (DSHB), Pax7), Ncad (DSHB, MNCD2; DSHB, 6B3; Abcam, ab18203), Cad6B (DSHB, CCD6B-1), Ecad (BD Transduction Laboratories, 610181; DSHB, 8C2 (Choi and Gumbiner, 1989 (link)); DSHB, 7D6 (Gallin et al., 1983 (link))), β-catenin (Abcam, ab6301), and p120-catenin (Cell Signaling, 4989S) was performed as follows: Embryos were fixed in 4% paraformaldehyde made in phosphate buffer for 15–40 minutes at room temperature. All washes were performed in TBST + Ca2++ with 0.5% triton x-100. Blocking was performed with 10% donkey serum in the same buffer. The primary antibodies (1:5–1:10 for all hybridoma antibodies and 1:200–1:1000 for all others, see Table 1) were incubated in the TBST buffer from overnight to two days at 4°C and secondary antibodies (Alexa Fluor, ThermoFisher Scientific 1:500 to 1:1000) were applied in the same buffer for either three hours at room temperature or overnight at 4°C.

Rogers C.D., Sorrells L, & Bronner M.E. (2018). A catenin-dependent balance between N-cadherin and E-cadherin controls neuroectodermal cell fate choices. Mechanisms of development, 152, 44-56.

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