Cholesteatoma keratinocytes were cultured on 35 mm culture dishes. The cells were grown to 70-80% confluence and then placed in KSFM with control (DMSO), GW0742 (100 nM), or GSK0660 (5 μM). After 24 h of treatment, the cells were washed and isolated using cell lysis buffer (Beyotime Institute of Biotechnology, China) containing protease inhibitors. Equal amounts of total protein were separated on 8% SDS-PAGE and transferred to a PVDF membrane (100 V for 60 min). The membrane was incubated with the primary antibodies overnight at 4°C, followed by the secondary peroxidase-conjugated antibody for 1 h. The bands were visualized by enhanced chemiluminescence and exposure to ECL Hyperfilm (GE Healthcare). The densitometry of bands was quantified with NIH Image 1.63 software. The protein expression was normalized to the amount of beta-actin. The following primary antibodies were used: anti-phospho-PDK1, anti-protein kinase B (AKT), anti-phospho-AKT, anti-phospho-GSK3β, and anti-phospho-PTEN (all from Cell Signaling Technology, Danvers, MA, USA). Antibodies against β-actin, Cyclin D1, and PPAR β/δ were from Genetex, Inc. (Genetex, CA, USA).
Anti phospho pten
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-phospho-PTEN is a primary antibody that specifically recognizes the phosphorylated form of the PTEN (Phosphatase and Tensin Homolog) protein. PTEN is a tumor suppressor that plays a crucial role in the regulation of the PI3K/AKT signaling pathway. The phosphorylation of PTEN is an important post-translational modification that modulates its activity and subcellular localization.
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Lab products found in correlation
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti phospho pdk1, by Cell Signaling Technology (2 mentions)
Anti phospho gsk3β, by Cell Signaling Technology (2 mentions)
Tank blotter, by Bio-Rad (2 mentions)
Bradford s assay, by Bio-Rad (2 mentions)
Anti gapdh 6c5, by Merck Group (2 mentions)
Anti pten, by BD (2 mentions)
Anti phospho akt ser473 d9e, by Cell Signaling Technology (2 mentions)
Anti akt pan c67e7, by Cell Signaling Technology (2 mentions)
Anti tbx2 c 17, by Santa Cruz Biotechnology (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Anti protein kinase b akt, by Cell Signaling Technology (1 mentions)
Hyperfilm ecl, by GE Healthcare (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Peroxide solution, by Merck Group (1 mentions)
Luminol, by Merck Group (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Anti phospho iκbα, by Cell Signaling Technology (1 mentions)
Anti wnt3a, by Cell Signaling Technology (1 mentions)
Anti phospho lrp6, by Cell Signaling Technology (1 mentions)
7 protocols using anti phospho pten
1
Cholesteatoma Keratinocyte Signaling Pathways
2
Western Blot Analysis of PTEN, TBX2/3, and AKT
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Cell extracts were made by lysing PBS washed cell pellets in radio-immunoprecipitation assay buffer (RIPA) supplemented with protease inhibitors (Complete protease inhibitor, Roche Diagnostics). Following incubation on ice, clear lysates were obtained by centrifugation. Protein concentrations were determined by Bradford's assay (Bio-Rad). For each sample, 30 μg of protein was loaded on each gel. Proteins were transferred onto a PVDF membrane using a tank blotter (Bio-Rad). The membranes were then blocked with 5% milk and 1X Tris buffered saline plus Tween 20 (TBST) and incubated with primary antibody overnight at 4°C. Membranes were then washed with 1X TBST and incubated with the corresponding secondary antibody. Membranes were again washed with 1X TBST, incubated with chemiluminescent substrate according to manufacturer's protocol (SuperSignal, Pierce) and visualized by autoradiography. The antibodies used include anti-PTEN (559600, BD Pharmingen), anti-phospho PTEN (9554, Cell Signaling), anti-TBX2 (C-17, Santa Cruz Biotechnology), anti-TBX2 (gift of C. Goding, University of Oxford), anti-TBX3 (A-20, Santa Cruz Biotechnology), anti-AKT (pan, C67E7, Cell Signaling), anti-phospho-AKT (Ser 473 D9E, Cell Signaling) and anti-GAPDH (6C5, Millipore). At least three biological replicates were performed for each experiment.
3
Western Blot Analysis of Signaling Proteins
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The cells were lysed using RIPA lysate (1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate). Equal amounts (20 µg) of the samples were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. The following primary antibodies (Cell Signaling Technology, Boston, MA, USA) were incubated with the membrane at 4 °C overnight: anti-ubiquitin-C (1:1000 dilution); anti-Wnt-3a (1:1000); anti-Wnt-7a (1:2000); anti-phospho-LRP6 (1:1000); anti-phospho-IκBα (1:1000); anti-phospho-p65 (1:1000); anti-phospho-PTEN (1:500); anti-phospho-Akt (1:1000) and anti-β-action (1:2000) antibodies. Secondary antibodies (Cell Signaling Technology, Boston, MA, USA, 1:5000 dilution) were added and incubated with enhanced chemiluminescence (ECL) reagent (including Luminol and Peroxide solution, Millipore, Schwalbach, Germany). The chemiluminescence, which reflected the expression of proteins, was visualized by X-ray film.
4
Antibody Sourcing and Characterization
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Anti-NDRG1 antibody was generated as previously described [43 (link)]. Other antibodies were purchased as follows: anti-α-tubulin antibody was from Sigma-aldrich Co; anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-phospho-HER3, anti-Met, anti-phospho-Met, anti-PDGFRβ, anti-phospho-PDGFRβ, anti- IGF-1Rβ, anti-phospho-IGF-1Rβ, anti-ERK1/2, anti-phospho-ERK1/2, anti-AKT, anti-phospho-AKT (Thr308 and Ser473), anti-mTOR, anti-phospho-mTOR (Ser2448 and Ser2481), anti-Raptor, anti-Rictor, anti-S6K, anti-phospho-S6K(Thr389), anti-phospho-S6, anti-PTEN, anti-phospho-PTEN, anti-IRS-1, anti-PDK1, anti-PARP, anti-phospho-PDK1, anti-TSC-1, anti-TSC-2, and anti-phospho-4EBP-1 antibodies were from Cell Signaling Technology (Beverly, MA); anti-HER2 and anti-HER3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Trevigen Inc. (Gaithersburg, MD): anti-p27 antibody was from BD Biosciences (San Jose, CA): anti-cleaved PARP antibody was from Promega (Madison, WI): anti-phospho-HER2 antibody was from Millipore (Billerica, MA): anti-β-actin was from Abcam (Cambridge, UK).
5
Extraction and Characterization of Fucoidan
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The antibodies including anti-phospho-GSK3β, anti-GSK3β, and anti-phospho-PTEN were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-β-actin, anti-NFATc1, and anti-phospho-NFATc1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemical reagents used in this study were analytical grade and obtained from Sigma (Saint Louis, MO, USA). The fucoidan was provided by Hi-Q Marine Biotech International Ltd, Taipei, Taiwan) and prepared as described previously [36 (link)]. Briefly, dried Sargassum hemiphyllum was incubated with hot water and lyophilized followed by incubation of 95% ethanol for overnight and then glycolytic enzyme was added. The low molecular weight fucoidan with average molecular weight of 800 Da (92.1%) containing fucose 210.9 ± 3.3 μmol/g and sulfate 38.9 ± 0.4% (w/w) was obtained by passing through suitable molecular weight cut-off membranes. The fucoidan dissolved in distilled H2O was used for subsequent tests.
6
Cellular Signaling Pathway Analysis
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Dimetil sulfoxide (DMSO), Folin-Ciocalteu, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt, sodium acetate, HEPES minimum 99.5% titration, albumin from bovine serum (BSA), reduced glutathione (GSH), oxidized glutathione, tetramethylethylenediamine (TEMED), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), D-Manitol, K2HPO4, KH2PO4, Triton X-100, β-mercaptoethanol, anti-rabbit immunoglobulin (HRP peroxidase-linked antibody), and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrate (FCCP) were obtained from Sigma-Aldrich (São Paulo, SP, Brazil). SDS, acrylamide, bis-acrylamide, and hybond nitrocellulose were obtained from GE Healthcare Life Division (Uppsala, Sweden). Anti-phospho-p38 (Thr180/Tyr182) and total form, anti phospho-AKT (Thr308) and total form, anti-phospho PTEN, anti-phospho and total JNK1/2 (Thr183/Tyr185), anti-phospho ERK1/2 (Thr202/Tyr204) and anti-total-ERK1/2, and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA). Poly (ADP)-ribose polymerase (PARP) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Kit Caspase-Glo 3/7 was obtained from Promega (Madison, WI). All other reagents were commercial products of the highest purity grade available.
7
Western Blot Analysis of PTEN, TBX2/3, and AKT
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