Pp2a a

Manufactured by Cell Signaling Technology

Sourced in United Kingdom, United States

PP2A-A is a laboratory reagent produced by Cell Signaling Technology. It is a recombinant protein that serves as the structural subunit of the protein phosphatase 2A (PP2A) enzyme complex. PP2A-A plays a core role in the regulation of cellular signaling pathways by dephosphorylating target proteins.

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Anti-PP2A-A (6 citations) PP2A-A subunit (2 citations)

6 protocols using pp2a a

Protein Expression Analysis Protocol

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To analyze the protein, tissue samples, and cultured cells were dissolved using a RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) (Beyotime, Nantong, China). Consistently, 30 μg of protein was loaded in each lane, fractionated by SDS-PAGE, and transferred onto a PVDF membrane. Then, the membrane was incubated at 4 °C overnight with human-specific phospho-mTOR(Ser2448) (Cell Signaling, 5536), mTOR (Cell Signaling, 2983), p62 (Cell Signaling, 8025), LC3B (Cell Signaling, 3868), ATG5 (Cell Signaling, 9980), PP2AA (Cell Signaling, 2039), PP2AB (Cell Signaling, 4953), CD24 (Abcam, ab76514), phospho-AKT (Thr308) (Cell Signaling, 13038), AKT (Cell Signaling, 4685), ABCG2 (Cell Signaling, 42078), (Abcam, London, UK), β-actin (Cell Signaling, 4970), and GAPDH (Cell Signaling, 5174) antibodies. The results were visualized by a chemiluminescent detection system (Pierce ECL substrate western blot detection system, Thermo Scientific, IL, USA).

Lu S., Yao Y., Xu G., Zhou C., Zhang Y., Sun J., Jiang R., Shao Q, & Chen Y. (2018). CD24 regulates sorafenib resistance via activating autophagy in hepatocellular carcinoma. Cell Death & Disease, 9(6), 646.

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Small Cell Lung Cancer Tissue Microarray Immunohistochemistry

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Small cell lung cancer tissue microarrays (TMA) were from US Biomax Inc. (LC818). IHC staining was performed using standard techniques described previously (26 (link)) with antibodies against PP2A A (Cell Signaling Technology) in the Pathology/Solid tumor core, The City of Hope. Briefly, each TMA was reviewed and scored by two independent pathologists on a scale of 0 to 3: 0+, no staining, no expression; 1+, weak staining, low expression; 2+, moderate staining, moderate expression; and 3+, strong staining, high expression. The IHC staining of PP2A A was examined by a pathologist who assigned a score 0+ (no staining), 1+ (5%–80% of weak stained cells), 2+ (50%–90% of moderate stained cells), 3+ (70%–100% of strong stained cells). The number of tumor cores with stage I disease (I-IA-IB) was 39 and the pathologic score assigned was between 0+ and 3+. A total of 28 tumor cores were stage II (II-IIA-IIB) and 12 were stage III/IV (IIIA-IIIB-IV). Both groups had the same range of pathologic scores as the stage I group. The TMA slide specification sheet is included in Supplementary Table S1.

Mirzapoiazova T., Xiao G., Mambetsariev B., Nasser M.W., Miaou E., Singhal S.S., Srivastava S., Mambetsariev I., Nelson M.S., Nam A., Behal A., Atri P., Muschen M., Tissot F.L., Miser J., Kovach J.S., Sattler M., Batra S.K., Kulkarni P, & Salgia R. (2021). Protein Phosphatase 2A as a Therapeutic Target in Small Cell Lung Cancer. Molecular Cancer Therapeutics, 20(10), 1820-1835.

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Antibody Sources for Protein Analysis

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Various specific antibodies were procured from reputable sources (ELAV like RNA binding protein 1, also known as ELVAL1 (HUR) and NCL): Santa Cruz Biotechnology (Santa Cruz, CA, USA); PTEN, PP2A-A, PP2A-B, PP2A-C, NEDD4, FBW7, ITCH, USP4, HA or Flag: Cell Signaling Technology (Beverly, MA, USA); PHLPP1 and PHLPP2: Bethyl Laboratories (Montgomery, TX, USA); p63α: Genetex (Irvine, CA, USA); XIAP: BD Biosciences (San Jose, CA, USA); Anti-AUF1: Aviva (San Diego, CA, USA); USP8, β-Actin and GAPDH: Proteintech (Rosemont, IL, USA).

Li T., Huang M., Sun N., Hua X., Chen R., Xie Q., Huang S., Du M., Zhao Y., Lin Q., Xu J., Han X., Zhao Y., Tian Z., Zhang Y., Chen W., Shen X, & Huang C. (2024). Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation. Journal of Experimental & Clinical Cancer Research : CR, 43, 50.

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Western Blot Analysis of DNA Damage Signaling

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Whole cell and homogenized tumor tissues were lysed in NP-40 lysis buffer [50 mM Tris/HCl, pH 7.4, 150 mM NaCl and 1% Nonidet P40, supplemented with Complete Protease Inhibitor Cocktail tablets and PhosStop phosphatase inhibitors (Roche, Indianapolis, IN)] and prepared as previously described (26 (link)). Total cellular proteins (40 μg) were separated on 12% or 15% SDS/PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon).. The membrane was then blocked for 1 hour at room temperature in 5% (w/v) non-fat milk in TBS-Tween-20 and probed overnight with primary antibodies followed by anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) in blocking buffer for 1 h. Membranes were subsequently incubated in Immobilon Western Blot Chemiluminescent HRP Substrate (Millipore) and developed on biomax XAR film (Kodak). Primary antibodies were purchased from Cell Signaling Technology: γH2AX (Ser139), p-Wee1 (Ser 642), Wee1, p-cdc2 (Tyr15), p-BRCA1 (Ser1524), p-Chk1 (Ser345), p-Chk1 (Ser317), Chk-1, phospho-Chk2 (Thr68), PP2A-C, PP2A-A, cleaved caspase-3 (Asp175), cleaved PARP (Asp214), p-histone H3 (Ser10), p-ATR (Ser428), and p-(Ser) 14-3-3 binding motif.

Chang K.E., Wei B.R., Madigan J.P., Hall M.D., Simpson R.M., Zhuang Z, & Gottesman M.M. (2014). The Protein Phosphatase 2A Inhibitor LB100 Sensitizes Ovarian Carcinoma Cells to Cisplatin-Mediated Cytotoxicity. Molecular cancer therapeutics, 14(1), 90-100.

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Antibody Procurement and Usage Guide

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Antibodies were obtained from the indicated suppliers: anti-PME-1 (Santa Cruz Biotechnology, #sc-25278), PP2A-C (Cell Signaling Technology, #2038), vinculin (Sigma-Aldrich, #V9131), B55α (Cell Signaling Technology, #5689), PP2A-A (mouse monoclonal, gift from Prof. S. Dilworth), MAPKAPK2 (Cell Signaling Technology, #3042), phospho-MAPKAPK2 (Cell Signaling Technology, #3007), demethylated PP2A-C (Sigma-Aldrich, #05-577), SP1 (Cell Signaling Technology, #9389), Hsp90 (Cell Signaling Technology, #4877), RIPK1 (Cell Signaling Technology, #3493), and phospho-RIPK1 Ser320 (Cell Signaling Technology, #58274).

Guffens L., Derua R, & Janssens V. (2023). PME-1 sensitizes glioblastoma cells to oxidative stress-induced cell death by attenuating PP2A-B55α-mediated inactivation of MAPKAPK2-RIPK1 signaling. Cell Death Discovery, 9, 265.

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Melanoma Cell Line Characterization

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Primary human melanocytes HEMn were ordered from Thermo Fisher Scientific (#C-102-5C) and cultured according to the manufacturer's protocol. Primary patent derived melanoma lines MM029, MM034, MM047, MM057, and MM099 were from Dr. Jean-Christophe Marine laboratory. All other cell lines were ordered from ATCC and cultured with the suggested protocol. The antibodies against pATM-S1981 (#13050), pCHK2-T68 (#2661 and #2197), cleaved PARP (Asp214; #5625), pERK-T202/Y204 (#4370), ERK1/2 (#4696), pRSK-S380 (#9335), RSK1 (8408), cleaved caspase-3 (#9664), MYC (#18583), E2F1 (#3742), P21 (#2947), PP2A-B (the regulatory subunit of PP2A [PR55]; #4953), PP2A-A (#2041), PP2A-C (#2159), and PP5 (#2289) were ordered from Cell Signaling Technology. The antibodies against PP4-C (PPP4C; #PA5-96059) and PP6-C (PPP6C; #PA5-28919) were ordered from Thermo Fisher Scientific. The antibodies against CHK2 (#05-649), pATM-S1981 (#05-740), and γH2AX (Ser139; #06-636-I) were ordered from Sigma. The antibody against PARP1 (#39561) was ordered from Active Motif. The antibodies against PP1-Cα isoform (PPP1CA; #A300-904A), PP1-Cβ isoform (PPP1CB; #A300-905A), and PP1-Cγ isoform (PPP1CC, #A300-906A) were ordered from Bethyl. The antibodies against ATM (sc-377293) and GAPDH (sc-25778) were ordered from Santa Cruz Biotechnology.

Yue J., Vendramin R., Liu F., Lopez O., Valencia M.G., Gomes Dos Santos H., Gaidosh G., Beckedorff F., Blumenthal E., Speroni L., Nimer S.D., Marine J.C, & Shiekhattar R. (2020). Targeted chemotherapy overcomes drug resistance in melanoma. Genes & Development, 34(9-10), 637-649.

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