Amyloid beta (Aβ) 1–42 and scrambled Aβ peptide were from rPeptide. Dimethyl sulfoxide (DMSO) was from Fisher Scientific. Okadaic acid was from Sigma. The antibodies used in this study were purchased from Santa Cruz Biotechnology (anti-PSD95), GenScript Corporation (anti-Gapdh), Millipore (anti-puromycin), Abcam (anti-synapsin-I, anti-PSD95, anti-p-eIF4E and anti-MAP2) and Cell Signaling (anti-eIF2A, anti-p-eIF2A, anti-eIF4E, anti-PDI, anti-FMRP, anti-eEF2, anti-p-eEF2, anti-PP1, anti-PP2A-A, anti-PP2A-B, and anti-PP2A-C). HRP-conjugated secondary antibodies were from Cell Signaling and Jackson ImmunoResearch.
Anti pp2ac
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-PP2Ac is a reagent that specifically binds and detects the catalytic subunit of Protein Phosphatase 2A (PP2A). PP2A is a serine/threonine phosphatase that regulates various cellular processes. Anti-PP2Ac can be used to study the expression and localization of PP2A in cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pp2a a, by Cell Signaling Technology (4 mentions)
Anti pp2a b, by Cell Signaling Technology (3 mentions)
Okadaic acid, by Merck Group (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Abmart (2 mentions)
Anti puromycin, by Abcam (1 mentions)
Anti synapsin 1, by Cell Signaling Technology (1 mentions)
Anti psd95, by Cell Signaling Technology (1 mentions)
Anti p eif4e, by Cell Signaling Technology (1 mentions)
Anti map2, by Cell Signaling Technology (1 mentions)
Anti eif2a, by Cell Signaling Technology (1 mentions)
Anti p eif2a, by Cell Signaling Technology (1 mentions)
Anti eif4e, by Cell Signaling Technology (1 mentions)
Anti pdi, by Cell Signaling Technology (1 mentions)
Anti fmrp, by Cell Signaling Technology (1 mentions)
Anti eef2, by Cell Signaling Technology (1 mentions)
Anti p eef2, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
10 protocols using anti pp2ac
1
Aβ Peptide Preparation and Antibody Analysis
2
Western Blotting Analysis of Signaling Pathways
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Whole‐cell lysates were prepared in M‐PER buffer supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), resolved by SDS/PAGE, and blotted with indicated antibodies. SuperSignal West Pico Chemiluminescent Substrate and SuperSignal Western Blot Enhancer (Thermo Fisher Scientific) were used to enhance blotting signal when needed. The following antibodies were used in this study: anti‐MAP4K4, anti‐phospho‐ERK1/2, anti‐phospho‐JNK, anti‐JNK, anti‐phospho‐p38, anti‐p38, anti‐AKT, anti‐phospho‐AKT, anti‐MEK1/2, anti‐phospho‐MEK1/2, anti‐RAS and anti‐PP2A‐C (Cell Signaling Technology, Danvers, MA, USA); anti‐ERK1/2, anti‐GAPDH, anti‐β‐actin, anti‐PP2A‐B56β, and anti‐MKP3 (Santa Cruz Biotechnology, Dallas, TX, USA); anti‐6Χ HIS (Immunology Consultants Laboratory, Inc., Lake Oswego, OR, USA). Erlotinib and trametinib were purchased from Selleck Chemicals (Houston, TX, USA). The protein phosphatase 2 (PP2A) inhibitor okadaic acid (OA) was purchased from Sigma Aldrich. The PP2A activator FTY720 was purchased from Cayman Chemical (Ann Arbor, MI, USA). MAP4K4 inhibitor PF‐06260933 was purchased from Tocris Bioscience (Avonmouth, Bristol, UK). EGF was obtained from Thermo Fisher Scientific.
3
Protein Extraction and Immunoprecipitation Protocol
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Cells were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland), as previously described [16 (link)]. Equal amounts of protein were separated by SDS–PAGE and transferred to membranes. For immunoprecipitation, cell lysates were incubated with antibodies and Protein A/G Magnetic Beads (HY-K0202, MedChem Express), followed by western blotting. Western blotting was performed as previously described [19 (link)] with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies, followed by visualization using chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel). The antibodies used were as follows: anti-cyclin G2 (DF2284, Affinity Biosciences), anti-Flag (M20008XS, Abmart), anti-PP2Ac (2038 T, Cell Signaling Technology), anti-STAT1 (14994S, Cell Signaling Technology), anti-p-STAT1 (Y701) (9167S, Cell Signaling Technology), anti-lamin B1 (sc-6216, Santa Cruz), anti-β-tubulin (M30109XS, Abmart), anti-GAPDH (M20006F, Abmart), and anti-IgG (3900S, Cell Signaling Technology).
4
Comprehensive Protein Analysis Protocol
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Anti-DDB1, Bethyl (IHC-0013401), IHC (1:400), IF (1:200); Anti-DDB1, Epitomics (3821-1), WB (1:10,000); Anti-DCAF1, Proteintech (11612-1-AP), IHC (1:200), IF (1:200), WB (1:1,000); Anti-MYC, Cell Signaling (2272), WB (1:1,000); Anti-HA, Cell Signaling (3724), WB (1:1,000); Anti-HA, Sigma (F3165), WB (1:1,000); Anti-ERK1/2, Santa Cruz (sc-94), WB (1:1,000); anti-FITC-α-tubulin, Sigma (F2168), IF(1:500); anti-CREST, Fitzgerald Industries International (90C-CS1058), IF (1:100); anti-TOP2B, Epitomics (3747-1), IF (1:200); anti-SMC3, Abcam (ab128919), IF (1:20); anti-MAD2, Abcam (ab9777), IF (1:100); anti-PP2A-A, Cell Signaling (2041), WB (1:1,000), IF (1:100); anti-PP2A-B, Cell Signaling (2290), WB (1:1,000); anti-PP2A-C, Cell Signaling (2259), WB (1:1,000); anti-NEDD8, Epitomics (1571-1), WB (1:1,000); anti-cyclin B, Cell Signaling (4138), WB (1:1,000); anti-securin, Abcam (ab3305), WB (1:1000); anti-CDC20, Santa Cruz (sc-8358), WB (1:500); anti-pCDK1(T161), Cell Signaling (9114), WB (1:1,000); anti-pERK1/2, Cell Signaling (9101), WB (1:1,000).
5
Protein Quantification and Western Blot Analysis
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BMDMs were lysed in 1×SDS sample buffer. Kidneys were lysed with RIPA buffer containing 1% NP-40, 0.1% SDS, 100 mg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma-Aldrich) on ice. The supernatants were collected after centrifugation at 16,000 g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay (BCA Protein Assay Kit, Pierce Thermo-Scientific, Rockford, IL) according to the manufacturer’s instruction. The primary antibodies were anti-PP2Ac (cat: 2038, Cell Signaling Technology, Boston, MA, USA, 1:1000), anti-PP2Acα (cat: ab106262, Abcam, Cambridge, UK, 1:1000), anti-PP2Acβ (cat: ab168371, Abcam, 1:1000), anti-methyl-PP2Ac (L309) (cat: ab66597, Abcam, 1:1000), anti-Itgb2 (cat: ab119830, Abcam, 1:1000), anti-Epac1 (cat: ab124162, Abcam, 1:1000), anti-FN (cat: F3648, Sigma-Aldrich, 1:10000), anti-Stat6 (cat: ab32520, Abcam, 1:1000), anti-p-Stat6 (T645) (cat: BS4186, Bioworld Technology, Nanjing, China, 1:1000), anti-Rap1a/b (cat: 4938, Cell Signaling Technology, 1:1000), anti-tubulin (cat: sc53646, Santa Cruz Biotechnology, 1:10000), and anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, 1:5000). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health ImageJ software package.
6
Multispecific Antibody Detection
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The following antibodies were used: anti-pSer19-MLC2 (3675), anti-Ki67 (12202), anti-cleaved caspase 3 (9661), anti-CD31 (77699), anti-pT696-MYPT1 (5163), anti-MYPT1 (2634), anti-pY461-Src (6943), anti-pT202/Y204ERK1/2 (4370), anti-ERK1/2 (4695), anti-pT308-Akt1 (2965), anti-pS473-Akt1 (4060), anti-Akt (2920), anti-PP2A-C (2038), anti-PP2A-A (2041), anti-Shc (2432), anti-PI3 kinase p85 (4257), and anti-RhoC (3430) (Cell Signaling Technology); anti-β-actin (A5316) (Sigma-Aldrich); anti-PyMT (sc-53,481), anti-GST (sc-138), anti-Src (sc-8056), and normal rat IgG (sc-2026) (SantaCruz Biotechnology); and anti-RhoA (ARH03) (Cytoskeleton, Inc.).
7
RhoA Activation and Protein Expression Assays
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The active form of RhoA was detected using the RhoA/Rac1/Cdc42 Activation Assay Combo Biochem kit (Cat.# BK030, Cytoskeleton Inc., Denver, CO, USA), following the manufacturer’s instructions. For western blotting, cell cultures were lysed with lysis buffer containing a protease and phosphatase inhibitor cocktail, and protein concentrations were determined using the Bradford assay. The proteins were then subjected to electrophoretic resolution and transferred to PVDF membranes. Western blotting was performed with the following antibodies: anti-PP2A−A, anti-PP2A−B, and anti-PP2A−C [Cat.# 9780T, Cell Signaling Technology (CST), Beverly, MA, USA], anti-Pcdh7 (Cat.# TA505452, Origene, Rockville, MD, USA), anti-phopho-GSK3β (Ser9) [Cat.# 9336, Cell Signaling Technology (CST), Beverly, MA, USA], anti-GSK3β [Cat.#, 9315; Cell Signaling Technology (CST), Beverly, MA, USA], and anti-actin (Cat.# sc-47778, Santacruz Biotechnology; Santa Cruz, CA, USA).
8
Western Blot Analysis of Cellular Signaling
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Cell pellets were lysed in radioimmunoprecipitation assay buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40, 1 mM dithiothreitol, 1 mM NaF, 1 mM sodium vanadate, 1 mM PMSF (Sigma-Aldrich, St Louis, MO, USA) and 1% protease inhibitors cocktail (Merck, Millipore, Kenilworth, NJ, USA). Protein extracts were quantitated and loaded on 8–12% SDS-polyacrylamide gel, electrophoresed and transferred to a polyvinylidene difluoride membrane (Millipore, Kenilworth, NJ, USA). The membrane was incubated with primary antibody, washed and incubated with horseradish peroxidase-conjugated secondary antibody (Pierce Biotechnology Inc., Rockford, IL, USA). Detection was performed using a chemiluminescent western detection kit (Cell Signaling Technology, Danvers, MA, USA). The antibodies used were anti-CIP2A, anti-phospho-Akt (Ser473), anti-Akt (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PP2Ac, anti-caspase-3, anti-caspase-9, anti-PARP (Cell Signaling Technology), anti-c-Myc (Proteintech Group, Inc, Rosemont, IL, USA) and anti-GAPDH (Abmart, Shanghai, China) antibodies.
9
Inhibitor Assay for Cell Culture
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For cell culture experiments, fetal bovine serum (FBS), penicillin-streptomycin, and Dulbecco's modified Eagle medium, low glucose (DMEM) were purchased from WelGENE (Gyeongsan-si, Gyeongsangbuk-do, Korea). 8-Br-cAMP was purchased from Sigma-Aldrich (St. Louis, MO, USA). Okadaic acid was purchased from Enzo (Farmingdale, NY, USA) and L-NAME was purchased from Abcam (Cambridge, MA, USA). The HDAC5 inhibitor TSA was purchased from Sigma-Aldrich (St. Louis, MI, USA). U73122 and BAPTA-AM were purchased from Calbiochem (La Jolla, CA, USA). Anti-iNOS was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, California, USA); anti-PP2Ac, anti-Epac1, antiphospho-CaMKII (Thr 286 ), and anti-CaMKII antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA), and anti-phospho-HDAC5 (Ser 498 ) and anti-HDAC5 antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-iNOS and anti-3-NT antibodies were purchased from Merck Millipore (Darmstadt, Hesse, Germany). DAF-FM diacetate was purchased from Molecular Probes (Eugene, OR, USA) and Fura-2 was purchased from Life Technologies (Grand Island, NY, USA). All other chemicals were of analytical grade.
10
Western Blot Analysis of Cellular Signaling
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