Pp2a c

Manufactured by Cell Signaling Technology

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PP2A-C is a recombinant protein that represents the catalytic subunit of the protein phosphatase 2A (PP2A) enzyme. PP2A is a serine/threonine-specific protein phosphatase that plays a critical role in the regulation of cellular processes by dephosphorylating its target proteins.

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Close spelling variants of this product

PP2A-c-α (2 citations) Anti-PP2A C subunit (2 citations)

17 protocols using pp2a c

Quantifying Hepatic Nuclear Proteins

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Nuclear proteins were extracted from livers with Nuclear Extraction Kit (Abcam, Cambridge, MA). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Waltham, MA). Western blot was performed as described previously [29] (link) to detect PPARα (peroxisome proliferator-activated receptor-α), CPT1 (Carnitine palmitoyltransferase I), SREBP (Sterol-regulatory element binding protein), ADPN receptor 1, ADPN receptor 2, methyl-PP2A-C(Santa Cruz Biotechnologies, Santa Cruz, CA), FAS (fatty acid synthetase), SCD1 (Stearoyl-CoA desaturase-1), PP2A (protein phosphatase 2A) A, PP2A B, PP2A C, pAMPK, LKB1 (Cell Signaling Technologies, Beverly, MA), and ChREBP (Novus Biologicals, Littleton, CO). Blots were scanned using a Bio-Rad Imaging System (Image Lab™ Upgrade for ChemiDoc™ XRS + System #170-8299). All specific bands were quantified with the Automated Digitizing System (Image Lab 4.1). Results are representative of three independent experiments.

Zhao C., Liu L., Liu Q., Li F., Zhang L., Zhu F., Shao T., Barve S., Chen Y., Li X., McClain C.J, & Feng W. (2019). Fibroblast growth factor 21 is required for the therapeutic effects of Lactobacillus rhamnosus GG against fructose-induced fatty liver in mice. Molecular Metabolism, 29, 145-157.

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Analyzing ATR Kinase Activation

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Cells were harvested by scraping or trypsinization and lysed with buffer [50 mM Tris–HCl, pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1X protease/phosphatase inhibitor cocktail (Thermo 1861280)]. 2X SDS loading buffer was added to the lysates before heating at 95°C for 5 min.
Three to eight percentage Tris-Acetate SDS PAGE gradient gels (Invitrogen EA0378) were used to resolve ATR-H from ATR-L; otherwise, standard SDS PAGE gels were used. PVDF membranes (Amersham 10061-492) were used to capture proteins transferred from gels. Chemiluminescent signal was captured using the GE Amersham Imager 680. Antibodies and their dilutions for western blots include pATR S428 (Cell Signaling 2853) 1:1000, PP2Ac (Cell Signaling 2038) 1:1000, GAPDH (Cell Signaling 5174) 1:1000, PARP 1 (Cell Signaling 9532) 1:1000, Bid (Cell Signaling 2002) 1:1000, or (Santa Cruz Biotechnology sc6538) 1: 500, Beta Actin (Invitrogen MA1-140) 1:5000, mtHSP70 (Invitrogen MA3-028) 1:1000, ATR (Bethyl Laboratories, Inc., A300-137A, A300-138A) 1:8000, ATRIP (ABclonal A7139) 1:1000, PP2A-Cα/β (Santa Cruz Biotechnology sc-80665) 1:500, Cleaved Caspase-3 (Cell Signaling 9664) 1:1000, PP4c (Bethyl Laboratories, Inc., A300-835A) 1:8000, PP5c (Bethyl Laboratories, Inc., A300-909A) 1:8000.

Makinwa Y., Cartwright B.M., Musich P.R., Li Z., Biswas H, & Zou Y. (2020). PP2A Regulates Phosphorylation-Dependent Isomerization of Cytoplasmic and Mitochondrial-Associated ATR by Pin1 in DNA Damage Responses. Frontiers in Cell and Developmental Biology, 8, 813.

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Renal Protein Expression Analysis

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Renal tissues or cells were harvested and centrifuged at 12,000 g to remove cell debris. Protein concentration was measured using a BCA assay kit (Pierce). Eighty micrograms of protein was subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After transfer, the membranes were blocked and blotted routinely with antibodies against VE-cadherin (BD Biosciences), α-SMA (Abcam), collagen1 (Abcam), PP2Ac (Cell Signaling Technology), occludin (Invitrogen), P-threonine/P-serine (Santa Cruz), nitrotyrosine (Cayman), acetylated-lysine (Cell Signaling Technology), phosphotyrosine (PY-20,Biolgend), PP2A,C subunit, demethylated (Millipore), vimentin (Boster) and CD31 (Proteintech). The immunolabeled proteins were detected by enhanced chemiluminescence (Pierce).The density of the bands was analyzed by Quantity One software (Bio-Rad).

Deng Y., Guo Y., Liu P., Zeng R., Ning Y., Pei G., Li Y., Chen M., Guo S., Li X., Han M, & Xu G. (2016). Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy. Scientific Reports, 6, 19821.

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Evaluation of SET Antagonist TD19 Effects

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SET antagonist TD19 (EMQA), a derivative of erlotinib with chemical name of N⁴-(3-ethynylphenyl)-6,7-dimethoxy-N²-(4-phenoxyphenyl) quinazoline-2,4-diamine), was kindly provided from Dr. Chung-Wai Shiau's lab. The chemical structure and synthesis of TD19 has been disclosed in previous studies [31 (link),33 (link)], and its mechanisms as a SET inhibitor has been also reported in prior studies [30 (link),32 (link)]. Akt inhibitor (MK-2206) inhibitor and ERK inhibitor (SCH772984) were purchased from Selleckchem (Houston, TX, USA). TD19 at different concentrations were dissolved in dimethyl sulfoxide (DMSO) and added to cells in Dulbecco's Modified Eagle Medium (DMEM) containing 1% FBS. Antibodies used for Western blots of pElk-1 and Elk were obtained from Santa Cruz Biotechnology (San Diego, CA, USA), Other such as CIP2A, PARP, PP2Ac, Akt, pAkt (Ser473), beta-actin, caspase-3, ERK, pERK, pBcl2 (Ser70), Bcl2, p c-Myc (S62), c-Myc, Lamin B, Tubulin, GFP and Myc-tag were purchased from Cell Signaling (Danvers, MA, USA). Cycloheximide (CHX) was purchased from Sigma-Aldrich (St Louis, MO, USA). MTT was purchased from Sigma-Aldrich.

Liu C.Y., Huang T.T., Chen Y.T., Chen J.L., Chu P.Y., Huang C.T., Wang W.L., Lau K.Y., Dai M.S., Shiau C.W, & Tseng L.M. (2019). Targeting SET to restore PP2A activity disrupts an oncogenic CIP2A-feedforward loop and impairs triple negative breast cancer progression. EBioMedicine, 40, 263-275.

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Immunofluorescence and Western Blotting Assays

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Antibodies raised against Hemagglutinin (Roche Cat# 3F10, RRID:AB_2314622 or Cell Signaling Technology Cat# 3724, RRID:AB_1549585), GRA1 (provided by J.-F- Dubremetz, UMR 5235 Centre National de la Recherche Scientifique, Montpellier, France), Toxofilin (provided by I. Tardieux, INSERM 1209, Grenoble, France), MIC2 (provided by D.Sibley, Washington University School of Medicine, St. Louis, MO), β-catenin (BD Biosciences Cat# 610153, RRID:AB_397554), GSK3β (Cell Signaling Technology Cat# 12456, RRID:AB_2636978), PP2A-B56 (#MABS270, Millipore), PP2A65 RA (Cell Signaling Technology Cat# 2039S, RRID:AB_10695607), PP2Ac (#2038, Cell signaling), Phospho-GSK3α/β (Cell Signaling Technology Cat# 8566S, RRID:AB_10860069), TBP (Abcam Cat# ab62126, RRID:AB_2287049), and TgQRS (van Rooyen et al., 2014 (link)) were used in the immunofluorescence assay and/or Western blotting. Immunofluorescence secondary antibodies were conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). Western blotting secondary antibodies conjugated to alkaline phosphatase was purchased from Promega. The inhibitors CHIR 99021(#252917-06-9) and BIO (#66746362–9) were purchased from R and D systems. Recombinant bacterial lipopolysaccharide (LPS) from Escherichia coli O26:B6 (Sigma-Aldrich) was used to stimulate the BMDMs.

He H., Brenier-Pinchart M.P., Braun L., Kraut A., Touquet B., Couté Y., Tardieux I., Hakimi M.A, & Bougdour A. (2018). Characterization of a Toxoplasma effector uncovers an alternative GSK3/β-catenin-regulatory pathway of inflammation. eLife, 7, e39887.

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Tau Kinase and Phosphatase Expression Profiling

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The changes in the expression and activation of tau kinases were examined using the following antibodies: GSK-3β (1:1000, BD Transduction Lab, Franklin Lakes, NJ) as well as the following antibodies purchased from Cell Signaling Technology, Inc. (Danvers, MA): phospho-GSK-3β (Ser9, 1:1000), p35/p25 (1:1000), phospho-Akt (Ser⁴⁷³, 1:1000), Akt (1:1000), SAPK/JNK (1:1000), phospho-SAPK-JNK (Thr¹⁸³/Tyr¹⁸⁵, 1:1000), p44/42 MAPK (Erk 1/2, 1:1000), phospho-p44/42 MAPK (Erk 1/2, Thr²⁰²/Tyr²⁰⁴, 1:1000), p38 MAPK (P38, 1:1000). Cdk5 (C-8), phospho-Ca²⁺/calmodulin-dependent protein kinases II (CaMKII, Thr²⁸⁶, 1:1000) and total CaMKII (1:1000) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-p38 (clone 6E5.2, p-P38, Thr¹⁸⁰/Tyr¹⁸², 1/1000) was purchased from Millipore, and GSK-3 α/β PY279/PY216 (Tyr²⁷⁹/Tyr²¹⁶, 1:1000) from Life Technologies (Grand Island, NY).
The changes in the expression of the tau phosphatases were determined using antibodies directed at PP2A-C (1:1000, Cell Signaling), PP5 (1:1000, Cell Signaling), PP2A-Bα subunit (2G9), 1:1000, Cell Signaling), demethylated PP2A-C subunit (4B7, 1:1000, Santa Cruz), PP2A-A subunit (1:1000, Cell Signaling), pan-calcineurin A (PP2B, 1:1000, Cell Signaling), and the PP1 catalytic subunit (E-9, 1:1000, Santa Cruz).

Whittington R.A., Virág L., Gratuze M., Petry F.R., Noël A., Poitras I., Truchetti G., Marcouiller F., Papon M.A., Khoury N.E., Wong K., Bretteville A., Morin F, & Planel E. (2015). Dexmedetomidine Increases Tau Phosphorylation Under Normothermic Conditions In Vivo and In Vitro. Neurobiology of aging, 36(8), 2414-2428.

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Quantitative Western Blot Analysis

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Western blotting was performed as described [14 (link)]. Primary antibodies against β-actin (A2066, Sigma), AKT (Cell Signaling, Beverly, MA, USA), phospho-AKT-Ser473 (Cell Signaling), PP2AC (Cell Signaling) and a secondary goat anti-rabbit antibody conjugated with horseradish peroxidase (GE Healthcare, Little Chalfont, UK) were used. Protein bands were visualized using Chemi-Lumi One L Western blotting substrate (Nacalai Tesque), and band intensities were measured using Image Lab software (Bio-Rad).

Shimura T., Sasatani M., Kamiya K., Kawai H., Inaba Y, & Kunugita N. (2015). Mitochondrial reactive oxygen species perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of PP2A in lowdose irradiated human fibroblasts. Oncotarget, 7(3), 3559-3570.

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Western Blotting of Cell Signaling Proteins

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Cell protein lysates were prepared, and Western blotting was performed according to our published protocols [34 (link), 105 (link), 107 (link)]. The following primary antibodies were used in this experiment: E2F1 (3742, Cell Signaling), E2F2 (sc-633, Santa Cruz Biotechnology), E2F3 (sc-878, Santa Cruz Biotechnology), cyclin B1 (sc-245, Santa Cruz Biotechnology), Mad2 (ab70383, Abcam), Bcl2 (2870, Cell Signaling), pBcl2 (Ser70) (2827, Cell Signaling), pSer/Thr/Phe (9631, Cell Signaling), cleaved caspase 3 (9661, Cell Signaling), cleaved PARP (5625, Cell Signaling), Hec1 (GTX70268, GeneTex, Irvine, CA), pBad (Ser136) (4366, Cell Signaling), Bad (9268, Cell Signaling), Mps1/TTK (3255, Cell Signaling), PP2Ac (2259, Cell Signaling), and Sgo1 (ab58023, Abcam). β-actin antibody (4970, Cell Signaling) was used as a loading control. For secondary antibodies, either goat anti-rabbit HRP (sc-2004) or goat anti-mouse HRP (sc-2005, Santa Cruz Biotechnology) were used. Signals were detected by using a Lumigen TMA-6 reagent (Lumigen Inc, Southfield, MI). Image J software (NIH, Bethesda, MD) was used to quantify protein levels.

Lee M., Rivera-Rivera Y., Moreno C.S, & Saavedra H.I. (2017). The E2F activators control multiple mitotic regulators and maintain genomic integrity through Sgo1 and BubR1. Oncotarget, 8(44), 77649-77672.

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Protein Extraction and Western Blot Analysis

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Cells were lysed in lysis buffer (50 mM HEPES, pH 7.5; 150 mM NaCl; 10% glycerol; 1% Triton X-100; 1.5 mM MgCl₂; 1 mM EGTA; 100 mM NaF; 10 mM pyrophosphate; 0.01% NaN₃, Complete, PhosSTOP, Roche), subjected to 7.5–15% SDS-PAGE, blotted, and incubated with antibodies (β-actin, #4970; caspase-3, #9665; CREB, #9197; phospho-CREB, #9198; GAPDH, #2118; IκBα #4814; phospho-p53, #9284; phospho-p65, #3033; PP2Ac, #2038—Cell Signaling; p53, 554293; poly(ADP-ribose)-polymerase, 551025—BD Biosciences, Heidelberg, Germany; p63, sc-843; p65, sc-8008—Santa Cruz; p73, PC385—Calbiochem, Darmstadt, Germany) and visualized by chemiluminescent detection (SuperSignal, Thermo Scientific). For immunoprecipitation, protein extracts were incubated with anti-p53 or -p65 antibody (#9282; #8242, Cell Signaling) and protein-A-Agarose (KPL, Gaithersburg, ML) beads overnight at 4 °C. Proteins were eluted and subjected to western blot analysis.

Müller I., Beissert S, & Kulms D. (2014). Anti-Apoptotic NF-κB and “Gain of Function” mutp53 in Concert Act Pro-Apoptotic in Response to UVB+IL-1 via Enhanced TNF Production. The Journal of Investigative Dermatology, 135(3), 851-860.

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Immunoblotting Analysis of Signaling Pathways

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Cells were harvested and protein was extracted using RIPA buffer (Sigma) with 1% v/v protease inhibitor cocktail (Sigma) and 1% v/v phosphatase inhibitor cocktail (Sigma). Samples were run on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes were then incubated overnight with the following primary antibodies: P-p44/42 MAPK, p44/42 MAPK, P-Akt (Ser473), Akt, PP2A-C, NNMT, PARP, Cleaved PARP, Caspase-3, Cleaved Caspase-3, ASK1, p38, P-p38, LCMT1, β-Actin (Cell Signaling Technology), and Me-PP2A-C (abcam). The membranes were then incubated for 1 hour with mouse or rabbit IgG-HRP conjugate secondary antibodies (Cell Signaling Technology) and developed.

Palanichamy K., Kanji S., Gordon N., Thirumoorthy K., Jacob J.R., Litzenberg K.T., Patel D, & Chakravarti A. (2016). NNMT silencing activates tumor suppressor PP2A, inactivates oncogenic STKs and inhibits tumor forming ability. Clinical cancer research : an official journal of the American Association for Cancer Research, 23(9), 2325-2334.

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