Cell and tissue lysates were prepared in RIPA lysis buffer using a dounce homogenizer as previously described [21 (link)]. Protein concentration was determined using Bradford reagent [22 (link)]. An aliquot of 30 to 50 μl of lysate was electrophoresd on a SDS-polyacrylamide gel. Western blotting was carried out as previously described [21 (link)]. Phospho-Akt at Ser473, Akt, PCNA, cyclin D1, HIF1/2-α and Caspase 3 antibodies were purchased from Cell Signaling Technology (Danvers, MA); PARP and GADPH antibodies were obtained from Santa Cruz Biotechnology. Expression of each protein was quantified by densitometry using National Institutes of Health image 1.62 software and normalized to a loading control.
Phospho akt at ser473
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Akt at Ser473 is a laboratory reagent produced by Cell Signaling Technology. It is used to detect and quantify the phosphorylation of the serine 473 residue of the Akt protein, a key regulator of cell growth, proliferation, and survival. This product can be utilized in various experimental techniques, such as western blotting, immunohistochemistry, and flow cytometry, to study the activation status of the Akt signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti hnrnp a2 b1, by OriGene (2 mentions)
Cmld 2, by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
Actinomycin d, by Merck Group (2 mentions)
Insulin elisa kit, by Mercodia (2 mentions)
Palmitic acid, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Hif1 2 α, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Antibody against β actin, by Merck Group (1 mentions)
Anti rac1, by Merck Group (1 mentions)
Anti nitrotyrosine antibody, by Merck Group (1 mentions)
Anti 3 nitrotyrosine antibody, by Abcam (1 mentions)
Tetramethylrhodamine tmrm, by Thermo Fisher Scientific (1 mentions)
Gw9662, by Merck Group (1 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Merck Group (1 mentions)
Anti h3k36me3, by Abcam (1 mentions)
7 protocols using phospho akt at ser473
1
Quantitative Western Blot Analysis of Cell Signaling
2
Antibody Immunodetection of Metabolic Proteins
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Antibodies against PPAR-γ, Akt1, phospho-Akt at Ser473 and VDAC were purchased from Cell Signaling (Danvers, MA). Antibodies against eNOS, p67phox, and p47 phox were purchased from BD Transduction laboratories (San Jose, CA). The anti-Rac1 and anti-phospho S617-eNOS were purchased from EMD Millipore (Temecula, CA). The anti-nitrotyrosine antibody was obtained from EMD Millipore (Billerica, MA). The anti-3-Nitrotyrosine antibody (cat#:ab110282) was obtained from Abcam (Cambridge, MA). The anti-CrAT was obtained from Proteintech (Rosemont, IL). The nitro-Tyr350 Akt1–specific antibody was raised against a synthetic peptide antigen CGRLPF(Y-NO2)NQDHEKL, where Y-NO2 represents 3-nitrotyrosine as described [33 (link)]. Antibody against β-actin, TGF- β1 and GW9662 were purchased from Sigma (St. Louis, MO). MitoTracker (cat#: M22425) and Tetramethylrhodamine (TMRM) were obtained from Invitrogen (Carlsbad, CA).
3
Antibody Immunoblotting for Protein Expression
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Antibodies were purchased from the following companies: anti-GLA from Santa Cruz; anti-PP1, Akt, phospho-PP1 at Thr320, phospho-Akt at Ser473, phospho-Akt at Thr308 from Cell Signaling Technology; anti-hnRNP A1 from Sigma; anti-hnRNP A2B1 from Acris; anti-SRp20 and SF2/ASF from Thermo Scientific; anti-H3K4me3, anti-H3K9me3, anti-H3K27me3, anti-H3K36me3, anti-H3K9me3, anti-H3K9A, anti-H3K9A, anti-H3K23A, anti-H3K27A, anti-H3S10P, anti-histone H3, anti-actin, and anti-HSP70 from Abcam; anti-p54nrb/NONO from Affinity Bioreagents; anti-PSIP1 from Bethyl Laboratories.
4
Antibody Characterization and Validation
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Antibodies were purchased from the following companies: anti-H3K27me1, H3K27me3, H3K79me1, H3K79me2, histone H3, SETD2, and tubulin from Abcam; anti-hnRNP A2/B1 from Acris; anti-SRPK1 and SRPK2 from BD Biosciences; anti-AKT, H3K27me2, PP1, phospho-PP1 at Thr320, phospho-AKT at Ser473, and cleaved-CASPASE-3 from Cell Signaling Technology; anti-H3K4me1, H3K4me2, H3K4me3, and H3K36me3 from Millipore; anti-pro CASPASE-3 from GeneTex; anti-hnRNP C1/C2, BAX, BCL-2, and PARP from Santa Cruz Biotechnology; anti-hnRNP A1, SRSF6, and TRA2B from Sigma; and anti-SRSF1 and SRSF3 from Zymed.
5
Evaluating Pharmacological Modulators in Cell and Animal Studies
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Bovine serum albumin (BSA, #B2064), oleic acid (OA, #O1008), palmitic acid (PA, #P0500), MG-132(#M7449), CQ (#C6628), actinomycin D (#SBR00013), and the TG assay kit (MAK266) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HuR-ARE interaction inhibitor CMLD-2 was purchased from Millipore (#5.38339.0001). The antibodies used were specific to HuR (Millipore, # 07–468), AKT (Cell Signaling Technology, #4691), phospho-AKT at Ser473 (Cell Signaling Technology, #4060), PTEN (Cell Signaling Technology, #9188), TUBA1A (Proteintech, #11224-1-AP), and GAPDH (Proteintech, # 60004–1). Lentiviruses encoding PTEN (Lenti-PTEN) or Lenti-LacZ were purchased from Jikai (Shanghai, China). Adenovirus expressing green fluorescent protein (AdGFP) and HuR (AdHuR) were purchased from Vigenebio (MD, USA). The insulin ELISA kit was purchased from Mercodia (10-1249-01). The assay kits used to measure serum ALT, AST, NEFAs, and TC were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
6
Platelet Signaling Pathway Analysis
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Human thrombin, PGE1, rhodamine-phalloidin, dimethyl sulfoxide (DMSO), ADP, fibrinogen, human fibrinogen, ferric chloride (FeCl3), Acetylsalicylic acid (ASA), carboxymethylcellulose (CMC), and all the reagents were purchased from Sigma (St. Louis, MO, USA). Equine tendon collagen (type I) and ATP luciferin/luciferase reagent were obtained from Chrono-log (Havertown, PA, USA). CRP was obtained from Dr Richard Farndale (Department of Biochemistry, University of Cambridge, UK). Phycoerythrin (PE)-conjugated isotype control IgGs, rat monoclonal antibodies against mouse P-selectin, activated αIIbβ3 (JON/A) were from Emfret Analytics (Eibelstadt, Germany). Antibodies against phospho-Syk at Tyr525/526 (Cat# 2710S), phospho-PLCγ2 at Tyr1217 (Cat# 3871S), phospho-PI3K p85α/β at Tyr458/p55α/γ at Tyr199 (Cat# 4228S), phospho-Akt at Ser473 (Cat# 9271S), phospho-ERK at Thr202/Tyr204 (Cat# 4337S), Total Syk (Cat# 12358S), Total PLCγ2 (Cat# 3872S), Total PI3K p85 (Cat# 4249S), Total Akt (Cat# 9272S), Total ERK (Cat# 9102S), and β-actin (Cat# 4967S) were obtained from Cell Signaling (Danvers, MA, USA). Monoclonal antibodies against phospho-integrin β3 at Tyr 759 and Total integrin β3 were obtained from Santa Cruz (Santa Cruz, CA, USA). Calcium dye (FLIPR Calcium Assay kit) was from Molecular Devices (Sunnyvale, CA, USA).
7
Investigating HuR-mediated regulation of lipid metabolism
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Bovine serum albumin (BSA, #B2064), oleic acid (OA, #O1008), palmitic acid (PA, #P0500), MG-132(#M7449), chloroquine (CQ, #C6628), actinomycin D (#SBR00013) and TG assay kit (MAK266) were purchased from Sigma. HuR-ARE interaction inhibitor CMLD-2 was purchased from Millipore (#5.38339.0001). The antibodies used were speci c for HuR (Millipore, #07-468), Akt (Cell Signaling Technology, #4691), phospho-Akt at Ser473 (Cell Signaling Technology, #4060), PTEN (Cell Signaling Technology, #9188), Tublin (Proteintech, #11224-1-AP), and GAPDH (Cell Signaling Technology, cat #2118). Lentivirus encoding PTEN (Lenti-PTEN) or Lenti-LacZ were from Jikai (Shanghai, China).
Adenovirus expressing GFP (AdGFP) and HuR (AdHuR) were from Vigenebio (MD, USA). The insulin ELISA kit was from Mercodia (10-1249-01). The assay kits used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonesteri ed fatty acids (NEFAs) and total cholesterol (TC) were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Adenovirus expressing GFP (AdGFP) and HuR (AdHuR) were from Vigenebio (MD, USA). The insulin ELISA kit was from Mercodia (10-1249-01). The assay kits used to measure serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonesteri ed fatty acids (NEFAs) and total cholesterol (TC) were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
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