Ab55676

Manufactured by Abcam

Sourced in United States

Ab55676 is a mouse monoclonal antibody that recognizes human Lamin A/C. This antibody can be used for various applications such as Western blot, immunofluorescence, and immunohistochemistry to detect Lamin A/C protein expression.

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5 protocols using ab55676

Western Blot Analysis of Protein Expression

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Cells were lysed with RIPA buffer (50mM Tris-HCl at pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM EDTA plus proteases and phosphatases inhibitors) for 1hr at 4 °C. Total protein quantification was performed with Bio-Rad Protein Assay Dye Reagent concentrate. A total of 50 μg of protein was separated on 15% SDS-PAGE gels at 100 V and transferred to PVDF membranes for 2 h at 200 mA. PVDF membranes were hybridized with mouse antibodies against L1CAM (HPA005830, Sigma-Aldrich), HIF1a (2015-1, Epitomics), GAPDH (ab9483, Abcam), NODAL (ab55676, Abcam), CXCR4 (ab124824, Abcam), pSmad2 (3108; Cell Signaling) and Smad2 (#5339; Cell Signaling), treated with peroxidase-conjugated goat anti-mouse or anti-rabbit Ig secondary antibody (DPVR-HRP, Immunologic), and then visualized by enhanced chemiluminescence (ECL Nova 2.0 XLS071, 2050 Cyanagen). N > 6. The antibodies used are listed in Supplementary Table S1.

Cave D.D., Hernando-Momblona X., Sevillano M., Minchiotti G, & Lonardo E. (2021). Nodal-induced L1CAM/CXCR4 subpopulation sustains tumor growth and metastasis in colorectal cancer derived organoids. Theranostics, 11(12), 5686-5699.

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Protein Expression Analysis by Western Blot

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Equal amounts of protein samples were extracted from cells and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% fat-free milk, then incubated with antibodies against ACE2 (1:3,000, ab108252, Abcam, US), VE-cadherin (1:1,000, ab33168, Abcam), EphA2 (1:1,000, 6997s, CST, USA), AKT (1:1,000, 4691, CST), p-AKT (1:2,000, 4060, CST), p38 (1:2,000, ab7952, Abcam), p-p38 (1:1,000, 4511, CST), Nodal (1:2,000, ab55676, Abcam), Notch4 (1:2,000, ab184742, Abcam), and Actin (1:2,000, ab8226, Abcam) overnight at 4°C, and the corresponding secondary antibodies (1:5,000, Thermo Fisher, USA) for 1 h at room temperature (RT). The blots were examined by an enhanced chemiluminescence detection kit (Millipore). Actin was used as a loading control, and all experiments were repeated three times independently. Expression quantification of proteins was performed using ImageJ software (National Institutes of Health, USA).

Xu K., Han H., Luo Y., Ye H., Lin H, & Ni L. (2021). The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation. Frontiers in Oncology, 11, 663671.

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Western Blot, Immunofluorescence, and Immunohistochemistry Antibodies

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Antibodies anti-human proteins used for Western blot, immunofluorescence and immunohistochemistry were the mouse monoclonal anti-Nodal (ab55676, Abcam, Cambridge, UK), anti-MAP2 (MS-249-R7, ThermoFisher Scientific), anti-PARP1 (sc-8007, Santa Cruz Biotechnology, Dallas, TX, USA), anti-CD44v6 (33-6700, ThermoFisher Scientific), anti-CD31 (M0823, Dako, Agilent Technologies, Santa Clara, CA, USA), anti-Glypican-2 (sc-393824, Santa Cruz Biotechnology), anti-Nucleolin (39-6400, ThermoFisher Scientific), anti-β-catenin (sc-7963, Santa Cruz Biotechnology), anti-LRP6 (sc-25317, Santa Cruz Biotechnology), anti-p53 (sc-126, Santa Cruz Biotechnology), and the rabbit polyclonal anti-N-Cadherin (04-1126, Millipore), anti-Ki-67 (ab833, Abcam), anti-P-Akt and anti-Akt (9271 and 9272, Cell Signaling Technology Inc., Danvers, MA, USA), anti-CD114 (PA5-28988, ThermoFisher Scientific), anti-PES1 (HPA066670, Sigma, St Louis, MO, USA), anti-NPM1 (HPA011384, Sigma), anti-Osteopontin (AB1870, Millipore).

Ognibene M, & Pezzolo A. (2020). Roniciclib down-regulates stemness and inhibits cell growth by inducing nucleolar stress in neuroblastoma. Scientific Reports, 10, 12902.

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NODAL Protein Expression Analysis in hPSCs

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Wildtype and NODAL-KO hPSCs were exposed to GSK3 inhibitor CHIR99021 (CHIR, 10 μM; Cayman Chemical) for 24 h to augment expression of NODAL protein before cells were lysed with RIPA buffer containing the cOmplete protease inhibitor cocktail (Roche). Protein concentration was determined by the Bradford assay using Protein Assay Dye Reagent Concentrate (Bio-Rad). An equal amount of protein (80 μg) from cell lysates of widetype and NODAL-KO hPSCs was resolved on 10% SDS-PAGE and transferred onto PVDF membranes (Thermo Fisher Scientific). Immunostaining was performed by blocking PVDF membranes with 5% skim milk for 1 h at room temperature followed by incubation overnight at 4 °C with mouse monoclonal antibodies to human NODAL (Abcam ab55676; 1:500 dilution) or human GAPDH (Sigma-Aldrich G8795; 1:20000 dilution) diluted in 5% skim milk. Membranes were washed with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) and stained with HRP-conjugated anti-mouse IgG antibody (Santa Cruz sc-516102; 1:3000 dilution) diluted in 5% skim milk for 1 h at room temperature. Protein bands were detected by a chemiluminescence assay using SuperSignal West Pico Plus and SuperSignal West Femto reagents (Thermo Fisher Scientific).

Zheng Y., Yan R.Z., Sun S., Kobayashi M., Xiang L., Yang R., Goedel A., Kang Y., Xue X., Esfahani S.N., Liu Y., Resto Irizarry A.M., Wu W., Li Y., Ji W., Niu Y., Chien K.R., Li T., Shioda T, & Fu J. (2022). Single-cell analysis of embryoids reveals lineage diversification roadmaps of early human development. Cell stem cell, 29(9), 1402-1419.e8.

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Immunohistochemical Analysis of Nodal Expression

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Four micron-thick, formalin-fixed, paraffin-embedded tissue sections on de-identified slides were used for IHC analysis of Nodal expression. IHC staining was performed using a Dako Plus autostainer (Dako, Inc, Carpinteria, CA, USA) as previously described. 19 In brief, following antigen retrieval and blocking steps, sections were incubated in a goat antihuman Nodal antibody (LS-B3955; LifeSpan Biosciences, Seattle, WA, USA, 1:150) for mouse xenografts and the mouse monoclonal anti-Nodal antibody (ab55676, Abcam, 1:200) for human tissue sections for 60 min, followed by appropriate biotinylated secondary antibody (Biocare Medical, Concord, CA, USA), and then streptavidin-horseradish peroxidase (Thermo Scientific Lab Vision, Fremont, CA, USA). Immunostaining was detected using either 3,3′-diaminobenzidine (Thermo Scientific Lab Vision) or 3-amino-9-ethylcarbazole (Biocare Medical) peroxidase chromogen substrates. Sections were counterstained with hematoxylin (Biocare Medical). As a negative control, adjacent serial sections were incubated with species appropriate irrelevant IgG (Jackson ImmunoResearch Labs) at the same concentration as primary antibodies. Sections were reviewed and scored as previously described. 27

Hendrix M.J., Kandela I., Mazar A.P., Seftor E.A., Seftor R.E., Margaryan N.V., Strizzi L., Murphy G.F., Long G.V, & Scolyer R.A. (2017). Targeting melanoma with front-line therapy does not abrogate Nodal-expressing tumor cells. Laboratory investigation; a journal of technical methods and pathology, 97(2).

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