Ab14592

Manufactured by Abcam

Sourced in United Kingdom

Ab14592 is a primary antibody that can be used for Western blotting, immunoprecipitation, and immunohistochemistry applications. It is an IgG1 antibody raised in mouse against a recombinant protein fragment corresponding to a region within amino acids 1 - 300 of human TRP53 (p53).

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Lab products found in correlation

9 protocols using ab14592

Western Blot Analysis of NEDD4, AKT, PTEN

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Cells were harvested and lysed with RIPA lysis buffer containing protease and phosphatase inhibitor (Thermo Scientific, Beijing, China). Protein concentration was measured using the BCA-kit, and 20 μg of each sample was resolved by SDS-PAGE. The primary antibodies included rabbit anti-human NEDD4 (Abcam, ab14592, UK), mouse anti-human AKT, rabbit anti-human p-AKT (Santa Cruz Biotechnology, sc-5298/135650, Dallas, TX, USA), rabbit anti-human PTEN (Abcam, ab32199, UK); mouse anti-human E-cadherin (Abcam, ab1416, UK), mouse anti-human Vimentin (Abcam, ab8979, UK) and mouse anti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Abcam, ab8245, UK). Optical densitometry analysis was performed using Image J software. GAPDH served as a loading control in each case.

Weng M., Luo Z.L., Wu X.L, & Zeng W.Z. (2017). The E3 ubiquitin ligase NEDD4 is translationally upregulated and facilitates pancreatic cancer. Oncotarget, 8(12), 20288-20296.

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Quantification of Free GFP Signals

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We used mouse monoclonal antibodies against GFP (clones 7.1 and 13.1; 11814460001; Roche), HA (clone F7; sc-7392; Santa Cruz Biotechnology, Inc.), anti-ubiquitin coupled to horseradish peroxidase (clone P4D1; sc-8017; Santa Cruz Biotechnology, Inc.) and rabbit polyclonal antibodies against 3-phosphoglycerate kinase (PGK; NE130/7S; Nordic-MUbio), Nedd4 (ab14592; Abcam), Bmh2 (a gift from S. Lemmon, University of Miami, Coral Gables, FL; Gelperin et al., 1995 (link)). Luminescence signals were acquired with the LAS-4000 imaging system (Fujifilm). Quantifications were performed using ImageJ (National Institutes of Health) on nonsaturated blots from independent experiments (n ≥ 3). The ratio of the free GFP signal over the total GFP signal in any given lane was calculated. A two-sided t test was performed and the p-values are indicated (NS, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Hovsepian J., Defenouillère Q., Albanèse V., Váchová L., Garcia C., Palková Z, & Léon S. (2017). Multilevel regulation of an α-arrestin by glucose depletion controls hexose transporter endocytosis. The Journal of Cell Biology, 216(6), 1811-1831.

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Cytokine-Responsive Protein Regulation

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Cytokines were obtained from Peprotech (Rocky Hill, NJ). S-Nitrosocysteine (CysNO) was synthesized by combining an equimolar concentration of L-cysteine with sodium nitrite in 0.2 N HCl and used within 1 h. We obtained antibodies against the following: galectin 1 (ab25138), NEDD4 (ab14592) and serpin B6 (ab97330) from Abcam (Cambridge, MA) (respective catalog numbers in parentheses). Tissue culture media and reagents were from Biological Industries (Beit Haemek, Israel). His-tagged proteins Trx(C35S) was expressed and purified from Escherichia coli as previously described [16 (link)]. Other materials were obtained from Sigma unless otherwise indicated.

Ben-Lulu S., Ziv T., Weisman-Shomer P, & Benhar M. (2017). Nitrosothiol-Trapping-Based Proteomic Analysis of S-Nitrosylation in Human Lung Carcinoma Cells. PLoS ONE, 12(1), e0169862.

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Protein Immunodetection by Western Blot

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Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. Antibodies used against PTEN were: (138G6, Cell Signaling), GFP45 (link), NEDD4-1 (ab14592, Abcam), p70S6K and phospho-p70S6k duet (8209, Cell Signaling), AKT (9272, Cell Signaling), phospho-AKT (4060, Cell Signaling) and actin (C-11, Santa Cruz Biotechnology). Immunocomplexes were visualized by fluorescently labeled secondary antibodies and detected using a PharosFX Plus molecular imager (Bio-Rad).

Nguyen H.N., Yang J.M., Miyamoto T., Itoh K., Rho E., Zhang Q., Inoue T., Devreotes P.N., Sesaki H, & Iijima M. (2015). Opening the conformation is a master switch for the dual localization and phosphatase activity of PTEN. Scientific Reports, 5, 12600.

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Antibody Validation for Cellular Analysis

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Goat polyclonal antibodies against Cx43 (AB1600), GST (AB9919-200), V5 (AB0096-500) and calnexin (AB0041-500) were obtained from SICGEN (Cantanhede, Portugal). Rabbit polyclonal antibodies against Cx43 (H-150) and mouse monoclonal antibodies against p62 (sc-28359) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit polyclonal antibodies against GABARAP (ab109364) and Nedd4 (ab14592) were obtained from Abcam (Cambridge, UK). Rabbit polyclonal antibodies against LC3B (PA1-16930) and mouse monoclonal antibodies against V5 (46-0705) were obtained from Invitrogen (Paisley, UK). Mouse monoclonal antibodies against ubiquitin (P4D1) were obtained from Covance (San Diego, CA, USA). Mouse monoclonal antibodies against EEA1 (610457) were obtained from BD Transduction Laboratories (San Jose, CA, USA). Rabbit polyclonal antibodies against ATG7 (A2856), Phorbol 12-myristate 13-acetate (PMA), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and cycloheximide (CHX) were obtained from Sigma (Saint Louis, MO, USA). Bafilomycin A1 was obtained from Bioaustralis (Smithfield, Australia).

Catarino S., Ribeiro-Rodrigues T.M., Sá Ferreira R., Ramalho J., Abert C., Martens S, & Girão H. (2020). A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins. Cells, 9(4), 902.

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Antibody Source and Characterization

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Antibodies against Calnexin (AB0041), Cx43 (AB0016; epitope within C-terminus of Cx43, aa. 237–382), GAPDH (AB0049) and GFP (AB0020) were purchased from SICGEN (Cantanhede, Portugal). Antibodies against Caveolin-1 (sc-894), Emerin (H12, sc-25284), Flotillin-1 (sc-25506), p21 (sc-756), HA (F-7, sc-7392), hnRNPA2B1 (sc-374053) and Importin-β/karyopherin-β1 (H-7; sc-137016) were obtained from SCBT (Heidelberg, Germany), and against Lamin B (NA12) were from ORP (San Diego, CA, USA). Antibodies against p53 (ab131442) and Nedd4 (ab14592) were from abcam (Cambridge, UK), against SART1 (H9092-BO1P) were from Abnova (Taipei City, Taiwan), and against ERp29 (HPA039456) were from Atlas Antibodies (Bromma, Sweden). Antibodies against SNW1 (AB_2619117), YBX1 (AB_2619228), HDAC1 (AB_2722192) and Lamin A/C (AB_2618203) were from DSHB (Iowa University, IA, USA). Antibodies against Cx43 (610062; epitope within C-terminus of Cx43, aa. 252–270) were from BD Biosciences (Franklin Lakes, NJ, USA), and against MYH7/β-MHC (A7564) were from ABclonal (Wuhan, China). Antibodies against myc (13-2500), Cx43 (710700; epitope within C-terminus of Cx43, aa. 237–382) and non-phosphorylated Cx43 (CX-1B1, 13–8300) were from Thermo Fisher Scientific (Waltham, MA, USA). Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA).

Martins-Marques T., Witschas K., Ribeiro I., Zuzarte M., Catarino S., Ribeiro-Rodrigues T., Caramelo F., Aasen T., Carreira I.M., Goncalves L., Leybaert L, & Girao H. (2023). Cx43 can form functional channels at the nuclear envelope and modulate gene expression in cardiac cells. Open Biology, 13(11), 230258.

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Protein Isolation and Western Blotting

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Protein isolation and western blotting were done as previously reported (26 (link)). Each protein band was normalized against β-actin. Anti-NEDD4 antibody (ab14592, Abcam; Cambridge, Cambridgeshire, United Kingdom) was used at 10,000:1 dilution, while anti-ALDH1A1 (ab134188) and anti-CD44 antibodies (ab157107, Abcam; Cambridge, Cambridgeshire, United Kingdom) as well as anti-β-actin antibody (A2228, Sigma Aldrich; St. Louis, MO, United States) were used at 1,000:1 dilution. The membrane was developed using a chemiluminescence detection system (ATTO Corporation, Tokyo, Japan).

Jeon S.A., Kim D.W., Lee D.B, & Cho J.Y. (2020). NEDD4 Plays Roles in the Maintenance of Breast Cancer Stem Cell Characteristics. Frontiers in Oncology, 10, 1680.

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JEV Infection Molecular Mechanisms

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Various commercially available antibodies were used in this study. Antibodies against LC3B (#3868), Beclin1 (#3495) and GAPDH (#2118) were obtained from Cell Signaling Technologies (Danvers, MA). The antibody against JEV NS3 (GTX125868) was from GeneTex (Irvine, CA). The antibody against Nedd4 (ab14592) was purchased from Abcam (Cambridge, MA). HRP-conjugated secondary antibodies against mouse (Cat #: 31430) or rabbit (Cat #: 31460) IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG (Cat #: A32723) and Alexa Fluor 555-conjugated goat anti-rabbit IgG (Cat #: A32732) were purchased from Life Technologies (Invitrogen^TM, Carlsbad, CA). Cyclosporin A (#S2286) and rapamycin (#S1039) were purchased from Selleck Chemicals (Houston, TX, USA). All of the plasmids used for JEV pseudotyped virus (JEVpv) generation were kindly provided by Yoshiharu Matsuura (Osaka University, Japan). A cDNA encoding the full-length human Nedd4 was cloned into the pcDNA3.1 vector. All constructs were confirmed by sequencing.

Xu Q., Zhu N., Chen S., Zhao P., Ren H., Zhu S., Tang H., Zhu Y, & Qi Z. (2017). E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells. Scientific Reports, 7, 45375.

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Characterization of Amyloid-Beta Oligomers

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Antibodies and reagents were obtained from the following commercial sources: The protein synthesis inhibitors cycloheximide (CHX) and anisomycin were purchased from Sigma-Aldrich and prepared in sterile water. All drugs were prepared in higher concentration stock solutions, stored in aliquots at −20°C, and thawed only once prior to use to preserve their potency. The GluA1 N-terminal mouse antibody (Millipore, MAB2263, RRID: AB 11212678) was purchased from Millipore, and GluA1 C-terminal rabbit antibody was homemade. Tubulin (Sigma-Aldrich, T3950, RRID: AB 477576) and GAPDH (Sigma-Aldrich, WH0002597M1, RRID: AB 1841 801) antibodies were purchased from Sigma-Aldrich. Ubiquitin (Abcam, ab7780, RRID: AB 306069), HA (Abcam, ab1424, RRID: AB 301017), USP46 (Abcam, ab88795, RRID: AB 2043162), and Nedd4 (Abcam, ab14592, RRID: AB 301364) antibodies were purchased from Abcam.
Synthetic Aβ_1–42 was purchased from Invitrogen and prepared according to the manufacturer’s instructions. Aβ oligomers were prepared as previously described [7 (link)]. In brief, the peptide was dissolved in phosphate-buffered saline (PBS) at 200 μM and incubated at 37°C for 24 h. Samples were aliquoted, stored at −20°C, and thawed once directly prior to use.

Zhang Y., Guo O., Huo Y., Wang G, & Man H.Y. (2018). Amyloid-β Induces AMPA Receptor Ubiquitination and Degradation in Primary Neurons and Human Brains of Alzheimer’s Disease. Journal of Alzheimer's disease : JAD, 62(4), 1789-1801.

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