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.
Anti akt pan c67e7
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Akt (pan) (C67E7) is a rabbit monoclonal antibody that recognizes all three isoforms of Akt (Akt1, Akt2, and Akt3). Akt is a serine/threonine protein kinase that plays a crucial role in various cellular processes, including cell growth, proliferation, survival, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho akt ser473 d9e, by Cell Signaling Technology (6 mentions)
Anti gapdh 6c5, by Merck Group (2 mentions)
Tank blotter, by Bio-Rad (2 mentions)
Complete protease inhibitor, by Roche (2 mentions)
Bradford s assay, by Bio-Rad (2 mentions)
Anti phospho pten, by Cell Signaling Technology (2 mentions)
Anti pten, by BD (2 mentions)
Anti tbx2 c 17, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated anti rabbit or anti mouse antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti nf κb p65 d14e12, by Cell Signaling Technology (1 mentions)
Anti phospho nf κb p65 ser536 93h1, by Cell Signaling Technology (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Anti fak, by Cell Signaling Technology (1 mentions)
Anti erk 137e5, by Cell Signaling Technology (1 mentions)
Anti phospho fak tyr925, by Cell Signaling Technology (1 mentions)
Ccd camera, by Fujifilm (1 mentions)
Anti α tubulin b 5 1 2, by Santa Cruz Biotechnology (1 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
10 protocols using anti akt pan c67e7
1
Western Blot Analysis of PTEN, TBX2/3, and AKT
2
Western Blot Analysis of Cell Signaling Pathways
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Cells (1×105) were washed once with PBS, lysed in 100 μl SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol, and 2% β-mercaptoethanol) and sonicated for 5 min. The samples were boiled for 5 min and subjected to SDS-PAGE, and the separated proteins were transferred onto a PVDF membrane (Merck Millipore) that had been incubated with 1:1,000-diluted primary antibody and then with HRP-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling), as recommended by the manufacturers. Primary antibody binding was detected using a Clarity™ Western ECL substrate (Bio Rad, Hercules, CA, USA), and images were captured by a CCD camera (Fuji Film, Tokyo, Japan). The following primary antibodies were used: anti-pERK (20G11, Cell Signaling), anti-ERK (137E5, Cell Signaling), anti-phospho-Akt (Thr308) (C31E5E, Cell Signaling), anti-Akt pan (C67E7, Cell Signaling), anti-phospho-FAK (Tyr925) (Cell Signaling), anti-FAK (Cell Signaling), anti-phospho-NF-κB p65 (Ser536) (93H1, Cell Signaling), anti-NF-κB p65 (D14E12, Cell Signaling), and anti-α-tubulin (B-5-1-2, Santa Cruz Biotechnology, Dallas, TX, USA).
3
Western Blot Analysis of Autophagy Markers
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Whole-cell lysates were resolved by SDS-PAGE and electrophoretically transferred to Hybond PVDF membranes (Amersham, GE Healthcare GmbH, Solingen, Germany). PVDF membranes were treated overnight with primary antibodies at 4 °C: anti-SQSTM1/p62 (#5114), anti-Beclin-1 (D40C5), anti-AMPKα (D5A2) (#5831), anti-cathepsin D (E179) (#69854), anti-phospho-Akt (Ser473) (D9E) (#4060), anti-Akt (pan) (C67E7) (#4691) were from Cell Signaling Technology (Danvers, MA, USA); anti-LC3B (L7543), and anti-BCL2 (BCL2-100) (B3170) were from Sigma–Aldrich (Merck KGaA); anti-human LAMP-1 (611042) (clone 25/Lamp-1) was obtained from BD Biosciences (Franklin Lakes, NJ, USA); anti-phospho-Beclin-1 (Ser295) (#PA5-35394) and anti-PBS TFEB (#PA1-9109) were obtained from Invitrogen (ThermoFisher Scientific Waltham, MA, USA). A mouse monoclonal anti-GAPDH antibody (6C5) (MAB-10578) (Immunological Sciences, Roma, Italy) was used as the loading control. After washing, PVDF membranes were incubated with the appropriate secondary antibody conjugate with horseradish peroxidase at room temperature for 1 h. Detection by chemiluminescence and analysis were performed as previously described [12 (link)].
4
p53 Epitope Peptide Analysis
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HLA-A2.1 restricted p53aa264-272 (LLGRNSFEV) was synthesized by Biosyntan (Berlin, Germany) and dissolved in DMSO. Antibodies used for Western blotting were mouse monoclonal antibodies (mAb) antihuman p53 (DO-2) sc-53394 (Santa Cruz Biotechnology, Heidelberg, Germany), antihuman p53 (HR231) MA1-12648 (Invitrogen), sheep antihuman p53 isoforms KJC12 (gift from Bourdon, Dundee, UK), rabbit mAbs anti-tri-methyl-Histone H3 (Lys4) (C42D8), anti-tri-methyl-Histone H3 (Lys9) (D4W1U) (Cell Signaling Technology), anti-HIF1a (Ab92498) (Abcam), rabbit mAb anti-Akt (pan) (C67E7) (Cell Signaling Technology, Cambridge), rabbit mAb anti-p21 (ab109520) (Abcam) and the mouse mAb anti-MDM2 (SMP14) sc-965 (Santa Cruz). Bafilomycin A1 (InvivoGen) was used as autophagy blocker.
5
Immunoblotting Protein Analysis Protocol
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For immunoblotting, cells were lysed in lysis buffer with protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration in samples was quantified by BCA assay (Thermo Fisher Scientific) before loading the samples for electrophoresis and membrane transfer. The transferred membrane was blocked with TBST (0.1% Tween 20) containing 5% BSA for 1 h at room temperature. The membrane was incubated with primary antibodies overnight including anti-Cleaved Caspase 3 (9662S; Cell Signaling), anti-p-AKT (Ser473, D9E; Cell Signaling), anti-AKT (pan, C67E7; Cell Signaling), anti-p-S6 (Ser235/Ser236, D57.2.2E; Cell Signaling), anti-PP2A Cα/β (F-8; Santa Cruz) and anti-β-actin (clone: 13E5; Sigma-Aldrich). Then, the membrane was washed and incubated with the corresponding secondary antibody for subsequent enhanced chemiluminescence (ECL; Thermo Fisher) exposure. The band intensity of all the immunoblot was analyzed by ImageJ software.
6
Antibody-Based Protein Expression Analysis
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The following antibodies and reagents were used in this study: anti-phospho-Akt (Ser473) (D9E) (1:2000), anti-Akt (pan) (C67E7) (1:1000), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, D13.14.4E) (1:2000), and anti-total p44/42 MAPK (ERK1/2) (1:1000) antibodies from Cell Signaling Technology (Boston, MA, USA); rabbit polyclonal anti-caveolin-1 (1:7500), anti-GAPDH (1:10,000) from Sigma-Aldrich (St. Louis, MO, USA); anti-rabbit IgG-Peroxidase antibody and resazurin sodium salt were obtained from Sigma-Aldrich (St. Louis, MO, USA); suramin hexasodium salt was obtained from Tocris Bioscience (Ellisville, MO, USA). Control (SC108080) and human caveolin-1 (SC29241) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents, unless mentioned, were obtained from Sigma-Aldrich (St. Louis, MO, USA).
7
Western Blot Analysis of PTEN, TBX2/3, and AKT
8
Assaying Prostate Cancer Cell Markers
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Dihydrotestosterone (DHT) and anti-androgen bicalutamide were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). Recombinant human EGF, FGF-2, FGF-7, FGF-10, hepatocyte growth factor (HGF), IGF-1, transforming growth factor (TGF) β1, vascular endothelial growth factor (VEGF), and IL-6 were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Rabbit polyclonal anti-PSA and mouse monoclonal anti-neuron-specific enolase (NSE; BBS/NC/V1-H14) antibodies were purchased from Dako Cytomation (Copenhagen, Denmark). Rabbit polyclonal anti-AR (N-20) and anti-EGFR (1005) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-CD31 and anti-Ki-67 and rabbit monoclonal anti-AR splice variant 7 (AR-V7; EPR15656) antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Rabbit monoclonal anti-phospho-STAT3 (Tyr705) (D3A7), anti-STAT3 (D3Z2G), anti-phospho-Akt (Ser473) (D9E), and anti-Akt (pan) (C67E7) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Rabbit polyclonal anti-p44/42 MAPK and mouse monoclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) (E10) antibodies were purchased from Cell Signaling Technology, Inc. Mouse monoclonal anti-β-actin (AC-15) antibodies were purchased from Sigma-Aldrich Co., LLC.
9
Western Blot Analysis of Protein Targets
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Cells were lysed in Laemmli buffer, proteins were separated on SDS-PAGE, and transferred to polyvinylidene fluoride membranes (Bio-Rad). Primary antibodies used were anti-FLAG (2368; NEB; 1:1000 dilution), anti-HA (3724; NEB; 1:1000 dilution), anti-mTOR (7C10; Cell Signaling; 1:1000 dilution), anti-DEPTOR (D9F5; Cell Signaling; 1:1000 dilution), anti-S6 (54D2; Cell Signaling; 1:1000 dilution), anti-phospho-S6S240/244 (2215; Cell Signaling; 1:1000 dilution), anti-pan-AKT (C67E7; Cell Signaling; 1:1000), antiphospho-AKTS473 (D9E; Cell Signaling; 1:1000 dilution), anti-ACTIN (D6A8; Cell Signaling; 1:2000 dilution) and anti-GAPDH (D16H11; Cell Signaling; 1:2000 dilution), anti-4G10 (05-321; Millipore–Sigma; 1:1000 dilution), anti-GFP (2956; Cell Signaling; 1:1000 dilution), antiubiquitin P4D1 (sc-8017; Santa Cruz; 1:1000 dilution), anti-EPHB2 D2X2I (83029; Cell Signaling; 1:1000 dilution), anti-EPHB2 (AF467; R&D Systems; 1:200 dilution), and anti-SYK D3ZE1 (13198; Cell Signaling; 1:500 dilution).
10
Taste Receptor Expression Analysis
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Caffeine (Sigma Aldrich, St. Louis, MO, USA) was dissolved in distilled water, Gemcitabine (Eli Lilly & Co., Indianapolis, IN, USA) in phosphate buffered saline (PBS; Sigma Aldrich), and 5-Fluorouracil (5-FU; Sigma Aldrich) in Dimethylsulfoxide (DMSO; Sigma Aldrich). For Western blot analysis the following antibodies were used: anti-ABCG2 (EPR2099, abcam); anti-Phospho-Akt Ser473 (D9E, Cell Signaling, Cambridge, UK), anti-pan-Akt (C67E7, Cell Signaling); anti-GAPDH (14C10, Cell signaling); anti-T2R10 (orb 164582; Biorbyt, Cambridge, UK); anti-tubulin (sc-58886, Santa Cruz); goat anti-rabbit IgG-horse radish peroxidase (HRP; Santa Cruz) as secondary antibody. For flow cytometry, a Fc Blocking Reagent (human, Miltenyi Biotec, Bergisch-Gladbach, Germany) was used; anti-T2R10 (ab138285, abcam, Cambridge, UK), a rabbit polyclonal isotype control (abcam), and anti-rabbit-IgG conjugated with phycoerythrin (PE) (Jackson Immunoresearch, West Grove, PA, USA).
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