Anti akt

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, China, Canada

Anti-Akt is a laboratory product that detects and measures the Akt protein. Akt is a key regulator of cellular processes such as growth, proliferation, and survival. Anti-Akt can be used to analyze the expression and activation of Akt in various experimental systems.

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Anti-p-Akt (96 citations) Anti-Akt (sc-8312, (7 citations) Anti-total Akt (6 citations) Anti-phosphorylated Akt (5 citations) Anti-Akt antibody (5 citations) Rabbit anti-human p-AKT (3 citations) Anti-Akt (sc-5298) (3 citations) Anti-AKT (Sc-1618, (2 citations) Anti-phospho-AKT (T308) (2 citations) Anti-Akt Ab (2 citations)

175 protocols using anti akt

Detailed Protein Analysis Methodology

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Chemicals and drugs of the purest available grade including NAC were provided by Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies anti-P-AKT (reacting with Ser473), anti-AKT, anti-Insulin-receptor (anti-IR), anti-fructokinase (anti-fructokinase), anti-glutathione-peroxidase (anti-GPx), and anti-glutathione reductase (anti-GR) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA; catalog number 6040S, 9272,sc-711, sc-50029, sc-133160, and sc-133245, respectively). Anti-COX-2 from CAYMAN Laboratories (Ann Arbor, MI, USA catalog number 160106), anti-iNOS, and anti-eNOS were obtained from Sigma (St. Louis, MO, USA; catalog number N7782). Anti-P-eNOS (Ser 1177) was provided by Cell Signaling Laboratory (Danvers, MA, USA; catalog number N3893); anti-glucokinase antibody (sheep anti-GST-glucokinase fusion protein antibody) was kindly provided by Dr. Mark Magnusson (Vanderbilt University, TN, USA). This antibody, another from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA; glucokinase-N-19: sc:1980), and anti-GAPDH from Millipore (Carlsbad, CA, USA; catalog number 92590) were also provided. Finally, a secondary antibody anti-rabbit IgG Peroxidase (developed in goats) was obtained from Sigma (St. Louis, MO, USA; catalog number A9169).

Castro M.C., Villagarcía H.G., Di Sarli Gutiérrez L., Arbeláez L.G., Schinella G., Massa M.L, & Francini F. (2024). Akt Signaling and Nitric Oxide Synthase as Possible Mediators of the Protective Effect of N-acetyl-L-cysteine in Prediabetes Induced by Sucrose. International Journal of Molecular Sciences, 25(2), 1215.

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Western Blot Antibody Characterization

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Standard Western-blot assays were used as described previously^{59 (link)}. The following primary antibodies were used: anti-PPARα (Abcam, Cata# ab24509), anti-ACLY (Santa Cruz, Cata# sc-517267), anti-FASN (Santa Cruz, Cata# sc-48357), anti-ACSL1 (cell signaling, Cata# 4047 S), anti-ACSL5 (Santa Cruz, Cata# sc-365478), anti-STAT3 (Santa Cruz, Cata# sc-8019), anti-pSTAT3 (cell signaling, Cata# 9145 S), anti-STAT1 (cell signaling, Cata# 14994 S), anti-pSTAT1 (cell signaling, Cata# 9177 S), anti-STAT4 (cell signaling, Cata# 2653 S), anti-pSTAT4 (cell signaling, Cata# 4134 S), anti-AKT (Santa Cruz, Cata# sc-5298, 1:2000 dilution), anti-pAKT (cell signaling, Cata# 9018 S), anti-pERK (cell signaling, Cata# 4376), anti-ERK (cell signaling, Cata# 9102), anti-pMAPK (cell signaling, Cata# 4511), anti-MAPK (cell signaling, Cata# 9212) and anti-β-actin (Sigma, Cata# A5441, 1:100,000 dilution) antibodies. Other than anti-AKT and anti-β-actin antibodies, the dilution for all other antibodies was 1:1000.

Yang X., Wang J., Chang C.Y., Zhou F., Liu J., Xu H., Ibrahim M., Gomez M., Guo G.L., Liu H., Zong W.X., Wondisford F.E., Su X., White E., Feng Z, & Hu W. (2024). Leukemia inhibitory factor suppresses hepatic de novo lipogenesis and induces cachexia in mice. Nature Communications, 15, 627.

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Fucoidan Extraction and Characterization

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Fucoidan from Fucus vesiculosus was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) as fucoidan. Fucoidan from Laminaria japonica was a gift from Hi-Q Marine Biotech International, Ltd. (Taiwan) as HiQ-fucoidan. PI, NAC, anti-actin (AC-74), anti-pERK1/2 (MAPK-YT) and anti-p21 (CP74) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Lipofectamine 2000 was purchased from Invitrogen (Grand Island, NY, USA). Anti-AKT, anti-p-AKT (S473) and anti-mouse IgG-HRP were purchased from Santa Cruz Biotechnology (CA, USA). Anti-CHOP, anti-GRP78, anti-eIF2α, and anti-rabbit IgG-HRP were purchased from GeneTex, Inc. (Hsinchu, Taiwan). Anti-caspase3 (8G10), anti-p-PERK (T980; 16F8), anti-PERK (D11A8), anti-p-eIF2α (Ser51, 119A11), anti-TLR4 (D8L5W) and anti-ATF4 (D4B8) were purchased from Cell Signaling (Beverly, MA, USA).

Hsu H.Y., Lin T.Y., Lu M.K., Leng P.J., Tsao S.M, & Wu Y.C. (2017). Fucoidan induces Toll-like receptor 4-regulated reactive oxygen species and promotes endoplasmic reticulum stress-mediated apoptosis in lung cancer. Scientific Reports, 7, 44990.

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Protein Extraction and Western Blotting

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A total protein extraction kit (Millipore, Billerica, MA, United States) was used to obtain total protein from atrial tissues. BCA working solution (Thermo Fisher Scientific, MA, United States) was used to determine protein concentrations. Bromophenol blue was added, and the samples were boiled to denature the protein. Proteins were then separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk powder for 1.5 h and then incubated overnight at 4°C with the following primary antibodies: anti-NPR-A [1:1000], anti-VASP [1:1000], anti-p-VASP [(Ser 239) 1:1000], anti-Akt [1:1000], anti-p-Akt [(Thr 308) 1:1000], anti-GSK-3β [1:1000], anti-p-GSK-3β [ (Ser 9) 1:1000], and anti-β-actin [1:5000]; all from Santa Cruz Biotechnology, Santa Cruz (CA, United States). The membranes were then washed with TBST and incubated with HRP-secondary antibodies at room temperature for 1.5 h. Finally, images were acquired using enhanced chemiluminescence solution.

Cao S., Han C., Xuan C., Li X., Wen J, & Xu D. (2022). Effects of cGMP/Akt/GSK-3β signaling pathway on atrial natriuretic peptide secretion in rabbits with rapid atrial pacing. Frontiers in Physiology, 13, 861981.

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Investigating SKA1 and Apoptosis Pathways

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TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The First Strand cDNA Synthesis kit and SYBR Premix Taq were from Takara (Dalian, Liaoning, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), bromodeoxyuridine (BrdU) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). DAPI, BCA protein assay and ECL Plus kits were obtained from Beyotime Institute of Biotechnology (Beijing, China). BD BioCoat Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA).
The primary antibodies against human SKA1 and cleaved caspase-3 were obtained from Abcam (Cambridge, MA, USA). Anti-Bcl-2, anti-Bax, anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt, anti-p21, anti-cyclin D1 and anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Biotinylated- and Cy3-conjugated anti-rabbit secondary antibodies were purchased form Boster (Wuhan, Hubei, China).

SHEN L., YANG M., LIN Q., ZHANG Z., MIAO C, & ZHU B. (2016). SKA1 regulates the metastasis and cisplatin resistance of non-small cell lung cancer. Oncology Reports, 35(5), 2561-2568.

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Western Blot Analysis of Protein Expression

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Lysates were prepared from frozen samples and cultured cells using the RIPA lysis buffer (Beyotime Institute of Biotechnology, Nantong, China). Membranes were incubated with the primary antibodies. For western blot analysis, we used the following antibodies: anti-PFKFB3 (Abcam; 1:3,000 dilution), anti-AKT (Santa Cruz Biotechnology; 1:2000 dilution), anti-ERK (Santa Cruz Biotechnology; 1:500 dilution), anti-E-cadherin (Santa Cruz Biotechnology; 1:500 dilution), anti-N-cadherin (Santa Cruz Biotechnology; 1:500 dilution),anti-Flotillin-1 (1:1000, Santa Cruz Biotechnology), anti-CD63 (1:1000, Sangon Biotech, Shanghai, China), and anti-CD9 (1:1000, Sangon Biotech, Shanghai, China), and mouse anti-β-actin (Santa Cruz Biotechnology; 1:2500 dilution) as a loading control. The expression levels of each protein were normalized by β-actin. The maximum intensity of each band was quantified using Image J software. The values are representative of at least three independent experiments.

Gu M., Li L., Zhang Z., Chen J., Zhang W., Zhang J., Han L., Tang M., You B., Zhang Q, & You Y. (2017). PFKFB3 promotes proliferation, migration and angiogenesis in nasopharyngeal carcinoma. Journal of Cancer, 8(18), 3887-3896.

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Profiling Signaling Protein Phosphorylation

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Cells were lysed in prechilled RIPA lysis buffer (Pierce Biotechnology, Rockford, IL) containing protease inhibitor cocktail (Roche, Basel, Switzerland). For detection of phosphorylation in signaling proteins, cells were serum deprived for 16 h, then stimulated with 20% fetal bovine serum (FBS) for 10 min, and phosphatase inhibitor cocktail (Roche) was added to the lysis buffer. Western blot was carried out as previously described with appropriate antibodies [14 (link)], including anti-LAPTM4B-N10-pAb (produced by our lab); anti-Bcl-2, anti-Bax, anti-phospho-tyrosine protein, anti-actin (all from Santa Cruz Biotechnology); anti-Akt, anti-cyclinD1, anti-phospho-p53 (Ser₁₅), anti-caspase3, anti-cleaved caspase3, and anti-phospho-Akt kinase (Ser₄₇₃) (all from Cell Signaling Technology).

Li M., Zhou R., Shan Y., Li L., Wang L, & Liu G. (2016). Targeting a novel cancer-driving protein (LAPTM4B-35) by a small molecule (ETS) to inhibit cancer growth and metastasis. Oncotarget, 7(36), 58531-58542.

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Protein Expression Analysis in Glioblastoma Cells

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U87 and T98G cells were lysed in radioimmunoprecipitation assay buffer [150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 8.0, 5.0 mM ethylenediaminetetraacetic acid, pH 8.0, 0.5 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride]. Protein concentrations were determined using the bicinchoninic acid method (Thermo Scientific, Rockford, IL, USA). Protein lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) by electroblotting. Primary antibodies for immunodetection were anti-CEP55 (Santa Cruz), anti-GLUT1 (Santa Cruz), anti-p-Akt^S473 (Santa Cruz), anti-p-Akt^T308(Santa Cruz), anti-Akt (Santa Cruz), anti-p-mTOR (Santa Cruz), anti-mTOR (Santa Cruz), anti-BAD (Santa Cruz), anti-caspase-9 (Santa Cruz), anti-GSK3-β (Santa Cruz), anti-p27 (Abcam) and anti-GAPDH (Santa Cruz). Subsequent to being incubated with Horseradish peroxidase (HRP) conjugated anti-rabbit or anti-mouse secondary antibodies (1: 10000, Santa Cruz) for 1 h, the immune complexes were detected using the enhanced chemiluminescence method.

Wang G., Liu M., Wang H., Yu S., Jiang Z., Sun J., Han K., Shen J., Zhu M., Lin Z., Jiang C, & Guo M. (2016). Centrosomal Protein of 55 Regulates Glucose Metabolism, Proliferation and Apoptosis of Glioma Cells via the Akt/mTOR Signaling Pathway. Journal of Cancer, 7(11), 1431-1440.

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Quantitative Western Blot Analysis

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Cell or tissue were lysed or homogenized in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, and 12 mM Na-deoxycholate supplemented with protease and phosphatase inhibitors cocktail. Nuclei and cytosol were obtained as previously described [26 (link)]. Protein concentration was determined by the Lowry protein assay. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes that were probed with the following antibodies: anti-nNOS (Santa Cruz), anti-SOD1 (Santa Cruz), anti-LDH (Santa Cruz), anti-poly-ADP-ribose polymerase 1 (PARP1), anti-actin (Santa Cruz), anti-tubulin (Sigma), anti-GSNOR (Thermo Scientific), anti-DJ-1 (Santa Cruz), anti-AKT (Santa Cruz), anti-phospho-AKT (Santa Cruz), anti-Nrf2 (Santa Cruz), and anti-H2B (Santa Cruz). After immunostaining with appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies, bands were revealed using the Amersham ECL detection system.

Rizza S., Cirotti C., Montagna C., Cardaci S., Consales C., Cozzolino M., Carrì M.T., Cecconi F, & Filomeni G. (2015). S-Nitrosoglutathione Reductase Plays Opposite Roles in SH-SY5Y Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis. Mediators of Inflammation, 2015, 536238.

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Western Blot Analysis of Notch Signaling

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Western blotting was performed as described previously (17 (link)). The primary antibodies and their sources were as follows: anti-Notch1, anti-Hes1, anti-NICD, anti-pERK, anti-ERK, and anti-pAKT (Cell Signaling Technology, Beverly, MA, USA); anti-Jagged1, anti-β-actin, anti-AKT, and anti-DUSP1 (Santa Cruz Biotechnology); anti-HBx (Merck-Millipore); anti-PTEN (Proteintech Group, Chicago, IL, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA).

Liao B., Zhou H., Liang H, & Li C. (2017). Regulation of ERK and AKT pathways by hepatitis B virus X protein via the Notch1 pathway in hepatocellular carcinoma. International Journal of Oncology, 51(5), 1449-1459.

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