Anti p foxo3a

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-p-FoxO3a is a laboratory reagent that detects the phosphorylated form of the FoxO3a transcription factor. FoxO3a is a member of the forkhead box O (FoxO) family of transcription factors that play a role in cellular processes such as apoptosis, cell cycle regulation, and glucose metabolism. The Anti-p-FoxO3a product specifically recognizes the phosphorylated form of FoxO3a, allowing researchers to study the activation and regulation of this important signaling pathway.

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10 protocols using anti p foxo3a

Molecular Mechanisms of Kidney Injury

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The following primary antibodies were used for immunoblot analysis or immunohistochemical staining: anti-KIM-1 (ab56015; Abcam, Cambridge, UK), anti-8-OHdG (MOG-100 P; JaICA, Shizuoka, Japan), anti-4-HHE (MHH-030n; JaICA), anti-MnSOD (ab16953; Abcam), anti-PI3K (610045; BD Transduction Laboratories, San Jose, CA, USA), anti-p-AKT (Ser473) (9271 S; Cell Signaling Technology, Danvers, MA, USA), anti-t-AKT (9272 S; Cell Signaling Technology), anti-p-FoxO3a (9466 S; Cell Signaling Technology), anti-t-FoxO3a (2497 S; Cell Signaling Technology), anti-β-actin (A5441; Sigma-Aldrich), anti-COX-IV (A301-899 A; Bethyl Laboratories, Montgomery, TX, USA), anti-Bcl-2 (sc-492; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax (DB005; Delta Biolabs, Gilroy, CA, USA), anti-active caspase-9 (9505 S; Cell Signaling Technology), and anti-active caspase-3 (AB3623; Millipore Corporation, St. Charles, MO, USA).

Lim S.W., Jin L., Luo K., Jin J., Shin Y.J., Hong S.Y, & Yang C.W. (2017). Klotho enhances FoxO3-mediated manganese superoxide dismutase expression by negatively regulating PI3K/AKT pathway during tacrolimus-induced oxidative stress. Cell Death & Disease, 8(8), e2972-.

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Immunoblotting of B Cell Signaling

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B cells cultured under various conditions were harvested and cell lysates were prepared in lysis buffer. The lysate was resolved on a 10% reducing SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. After blocking with Tris-buffered saline (pH 7.4) containing 5% dried skimmed milk, the membrane was incubated with antibodies, followed by probing with HRP-conjugated anti-rabbit or -mouse antibody (Sigma). The bands were detected by chemiluminescence with ECL detection reagents (Life Technologies). Antibody to Phospho-STAT6 (Tyr641, 56,554), anti-STAT6 (5,397), anti-p-PKA C(4,781), anti-PKA C-α (5,842), anti-p-CREB (9,198), anti-CREB (9,197), PI3 Kinase p110 δ 34,050, anti-p-Akt (Ser473, 4,060), anti-Akt (4,685), anti-p-FoxO1 (Ser256, 9,461), anti-FoxO1 (2,880), anti-p-FoxO3a (Ser253, 13,129), anti-FoxO3a (12,829), anti-PPARγ (2,443) were from Cell Signaling Technology (Danvers, MA, USA). Anti-ubiquitin (PTM-1106) was from PTM BIO (Beijing, China). Anti-β-Actin (66009-1-Ig) was from Proteintech.

Wu J., Wang Y., Zhou Y., Wang Y., Sun X., Zhao Y., Guan Y., Zhang Y, & Wang W. (2020). PPARγ as an E3 Ubiquitin-Ligase Impedes Phosphate-Stat6 Stability and Promotes Prostaglandins E2-Mediated Inhibition of IgE Production in Asthma. Frontiers in Immunology, 11, 1224.

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Western Blot Analysis of FoxO Proteins

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Western blotting was performed as described elsewhere [15 (link)]. Briefly, 10 μg cytoplasmic or nuclear protein was separated on SDS-PAGE and transferred to PVDF membranes. Membranes with nuclear protein were incubated with anti-FoxO3a (#12829) or anti-FoxO1 (#2880). Membranes with cytoplasmic protein were incubated with anti-p-FoxO3a (#2599), anti-p-FoxO1 (Ser256) (#9461), anti-p-AKT (Ser473) (#9271), anti-p-AKT (Thr308) (#4056) or anti-AKT (#9272; Cell Signaling Technology, Danvers, MA, USA) antibodies at 4°C overnight diluted 1:1000 in Solution 1 (TOYOBO, Co., Ltd., Osaka, Japan). After TBST washes, membranes were incubated with HRP-conjugated sheep anti-rabbit IgG (GE Healthcare Life Science, Piscataway, NJ, USA) diluted 1:2000 in Solution 2 at room temperature for 60 min. Internal controls were mouse anti-Hsc70 (sc-7298; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for cytoplasmic immunoblotting and mouse anti-Histone H3 (#4499) (Cell Signaling Technology, Danvers, MA, USA) for nuclear immunoblotting. Hsc70 and Histone H3 were used for normalization.

Zhu S., Tian Z., Torigoe D., Zhao J., Xie P., Sugizaki T., Sato M., Horiguchi H., Terada K., Kadomatsu T., Miyata K, & Oike Y. (2019). Aging- and obesity-related peri-muscular adipose tissue accelerates muscle atrophy. PLoS ONE, 14(8), e0221366.

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Comprehensive Antibody Validation Protocol

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The following rabbit antibodies used for Western blot, ChIP, IHC or IF were all from Cell Signaling Technology (1:1000): anti-SUMO1 (C9H1) mAb, anti-c-Myc (D84C1) mAb, anti-c-Met (D1C2) XP mAb, anti-Jun (60A8), anti-Bcl2 (#2872), anti-YY1 (13G10), anti-SP1 (D4C3), anti-HA-tag (C29F4), anti-FOXO3a (D19A7), and anti-p-FOXO3a (Ser253). Anti-CTCF, anti-Myb, anti-E2F3 (N2C3) and anti-CDK4 (GTX102993) were from GeneTex. Anti-E2F1 (KH95) and anti-GAPDH (I19) were from Santa Cruz Biotechnology. Rabbit anti-SAE2/UBA2 (1:1000) (ab58451), mouse anti-SAE2 (ab118404) and anti-SAE1 (ab185949) were from Abcam. Mouse monoclonal antibody against SUMO2/3 (1E7) was from MBL Medical & Biological Laboratories Co., Ltd. Rabbit anti-c-Myc (ab32072) from abcam was used for IHC to validate c-Myc expression in mouse xenograft tumor.

Li Y.J., Du L., Aldana-Masangkay G., Wang X., Urak R., Forman S.J., Rosen S.T, & Chen Y. (2018). Regulation of miR-34b/c-targeted gene expression program by SUMOylation. Nucleic Acids Research, 46(14), 7108-7123.

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Western Blot Analysis of Apoptosis Regulators

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Western blot analyses were performed according to the standard method as described previously (23 (link)), using anti-SphK1 antibody (Abgent, San Diego, CA, USA); anti- Akt, anti-p-Akt, anti-cleaved caspase 3, anti-PARP, anti-Bim, anti-bax, anti-Bcl-xL, anti-p-FoxO3a, and anti-Bcl-2 antibodies (Cell Signaling, Danvers, MA, USA). The membranes were stripped and re-blotted with an anti-α-tubulin Ab (Sigma, St. Louis, MO, USA) as a loading control.

XIONG H., WANG J., GUAN H., WU J., XU R., WANG M., RONG X., HUANG K., HUANG J., LIAO Q., FU Y, & YUAN J. (2014). SphK1 confers resistance to apoptosis in gastric cancer cells by downregulating Bim via stimulating Akt/FoxO3a signaling. Oncology Reports, 32(4), 1369-1373.

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Compound PD Mechanism of Action

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The test compound, PD (structure shown in Fig. 1A), was purchased from Must Bio-Technology Co., Ltd. (Chengdu, China). The purity of the test compound was determined to be greater than 98% by HPLC. All chemicals and solvents used were of analytical grade or of the highest grade available. Cell culture supplies, such as culture media and fetal bovine serum (FBS), were obtained from Hyclone (Carlsbad, CA, USA). The anti-human MDM2 antibody was obtained from Calbiochem (Billerica, MA, USA), the anti-FOXO3a, anti-p-FOXO3a and anti-p27 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the anti-human E2F1, CDK2, CDK4, CDK6, Bax and Bcl2 antibodies were obtained from Boster Biotechnology (Wuhan, China). Anti-human Cyclin D1, PARP, caspase8, caspase9 and caspase3 antibodies were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Antibodies against human p21 and p53 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against CDK1 and Cyclin B1 were obtained from Bioworld Technology (St. Louis Park, MN, USA). The GAPDH antibody (T5168) was from Goodhere Biotechnology (Hangzhou, China).

Zhou R., Lu Z., Liu K., Guo J., Liu J., Zhou Y., Yang J., Mi M, & Xu H. (2014). Platycodin D Induces Tumor Growth Arrest by Activating FOXO3a Expression in Prostate Cancer in vitro and in vivo. Current Cancer Drug Targets, 14(9), 860-871.

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Western Blot Analysis of Protein Expression

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Cells were rinsed in PBS and lysed in Pro-Prep lysis buffer (iNtRON Biotechnology, Seoul, South Korea) containing protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). The protein concentration was determined using a bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). Individual proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were probed with the appropriate primary antibodies diluted in PBS with Tween-20 containing 1% BSA and then with secondary horseradish peroxidase-conjugated antibodies. Bound antibodies were visualized using ECL reagents (Millipore). The following primary antibodies were used for western blotting: anti-IRα, anti-IRS-1, anti-p-mTOR, anti-mTOR, anti-pS6K, anti-S6K, anti-pAkt, anti-Akt, anti-p-ERK, anti-ERK, anti-Foxo3a, anti-pFoxo3a, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies from Cell Signaling Technology (Denver, MA, USA); anti-Atrogin-1 and anti-MuRF-1 antibodies from ECM Biosciences (Versailles, KY, USA); an anti-ubiquitin antibody from AbFrontier (Seoul, Korea); and an anti-α-tubulin antibody from Sigma-Aldrich (St. Louis, MO, USA).

Yoo W., Choi H., Son Y.H., Lee J., Jo S., Jung D., Kim Y.J., Koh S.S., Yang Y.R., Kwon E.S., Lee K.P., Noh K.H., Kim K.W., Ko Y., Jun E., Kim S.C, & Kim S. (2021). Pancreatic cancer induces muscle wasting by promoting the release of pancreatic adenocarcinoma upregulated factor. Experimental & Molecular Medicine, 53(3), 432-445.

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Immunoblot and Immunohistochemical Analysis of Signaling Pathways

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For immunoblot and immunohistochemical analysis, various antibodies like rabbit monoclonal anti-p-MAPK, anti-MAPK, anti-p-Akt (Ser & Thr) and anti-Akt, anti-p-GSK-3β, anti-GSK-3β, anti-p-FoxO3a, anti-FoxO3a monoclonal mouse anti-CD-31, rabbit anti-caspase 3/7 were purchased from Cell Signaling Technology, Beverly, MA, USA. Mouse anti-Ki-67, rabbit monoclonal anti-PARP, mouse monoclonal anti-Bcl-2, anti-Bax, anti-Mcl-1, anti-Bcl-xL, HRP conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse monoclonal anti-β-actin and other reagents were procured from Sigma Aldrich, St. Louis, MO, USA. Antibodies dilutions have been made as per the manufacturer’s instruction. The pcDNA3-Akt-HA plasmid was obtained as gift sample from Dr. Guy Salvesen (University of California, San Diego, CA, USA). siRNA Akt and pcDNA3.1(-) were purchased from Cell Signaling Technology, Beverly, MA, USA and Invitrogen, USA respectively. Opti-MEM^®I Reduced Serum Media and fetal bovine serum (FBS) were purchased from Gibco-BRL Invitrogen Corporation, CA, USA. FuGENE® HD transfection reagent was obtained from Roche Applied Science, Mannheim, Germany. Trypsin, Bovine serum albumin (BSA) and antibiotics (10,000 U/L penicillin and 10 mg/L streptomycin) were purchased from Himedia, Mumbai, India.

Dey G., Bharti R., Dhanarajan G., Das S., Dey K.K., Kumar B.N., Sen R, & Mandal M. (2015). Marine lipopeptide Iturin A inhibits Akt mediated GSK3β and FoxO3a signaling and triggers apoptosis in breast cancer. Scientific Reports, 5, 10316.

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Protein Quantification and Western Blotting

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Protein concentration was determined by Lowry's method with bovine serum albumin as a standard (9) . Proteins (10 µg / lane) obtained from cell homogenates were subjected to immunoblotting. Western blotting immunoprecipitation analysis was performed by the method described previously (7) . The following antibodies were used : anti-IRS-1 (Calbiochem, La Jolla, CA), anti-Cbl-b (no.9498, Cell Signaling Technology), anti-p-FOXO3a (#9466, Cell Signaling Technology), anti-FOXO3a (#2497, Cell Signaling Technology), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse IgG (M9044 ; Sigma), anti-rabbit IgG (no.7074, Cell Signaling Technology).

Kitahata K., Uchida T., Taniguchi R., Kato A., Sugiura K., Sakakibara I., Oarada M., Fukawa T., Junsoo P., Inho C, & Nikawa T. (2022). Additional effects of simultaneous treatment with C14-Cblin and celastrol on the clinorotation-induced rat L6 myotube atrophy. The journal of medical investigation : JMI, 69(1.2).

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Quantification of Oxidative Stress Markers

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Whole cell or tissue samples were homogenized in RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX) with protease inhibitor cocktail (Thermo Scientific) and 40 μg total protein was used for immunoblot analysis according to our previous protocols [27] . Blots were probed with anti-Klotho (1:200; Thermo Scientific), anti-3-nitrotyrosine (1:500; Abcam, Cambridge), anti-SOD2 (1:500; Santa Cruz Biotechnology), anti-RIP1 (1:100; Santa Cruz Biotechnology), anti-RIP3 (1:500; Santa Cruz Biotechnology), anti-IL-1 beta (1:1000; Abcam), anti-tubulin (1:1000; Abcam), anti-beta-actin (1:2000; Abcam), anti-catalase (1:1000; Abcam), anti-GPX4 (1:1000; Abcam), anti-t-FoxO1 (1:1000; Cell Signaling Technology, Beverly, CA), antip-FoxO1 (1:1000; Cell Signaling Technology), anti-t-FoxO3a (1:1000; Cell Signaling Technology), anti-p-FoxO3a (1:1000; Cell Signaling Technology), or anti-GAPDH (1:1000; Cell Signaling Technology) antibodies overnight at 4 °C. After washing, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000; Cell Signaling Technology). After washing, specific signals were determined using an ECL kit (Thermo Scientific) on a Tanon 5200 Chemiluminescent Imaging System (Tanon, Shanghai). Gray values were analyzed with ImageJ software.

Qian Y., Guo X., Che L., Guan X., Wu B., Lu R., Zhu M., Pang H., Yan Y., Ni Z, & Gu L. (2018). Klotho Reduces Necroptosis by Targeting Oxidative Stress Involved in Renal Ischemic-Reperfusion Injury. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 45(6).

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