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7 protocols using anti pt308 akt

1

Western Blot Protein Detection

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Total proteins were extracted using 1X cell lysing buffer (Cell Signaling Technology). Samples were separated by electrophoresis in SDS- polyacrylamide gels (12%) and transferred to PVDF membranes. Membranes were blocked for 1 h with PBST (PBS plus 0.1% Tween-20) containing 5% non-fat milk. Blots were incubated overnight at 4°C with primary antibodies in PBST containing 5% BSA at the manufacturer's recommended dilution. The antibodies were purchased from the following suppliers: FKBP4 (ProteinTech); anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pT286 Cyclin D1, anti-Cyclin D1, anti-E2F-1, anti-pT37/46 4E-BP1, anti-pS9 GSK-3β, anti-GSK-3β, and anti-mTOR (Cell Signaling Technology); anti-GAPDH and anti-Actin (Santa Cruz Biotechnology); anti-Tubulin and anti-PIK3R2 (Sigma-Aldrich). After washing, blots were incubated with either an anti-rabbit HRP, or an anti-mouse HRP-conjugated antibody (Cell Signaling Technology) for 1 h at 25°C. After washing, blots were developed with Supersignal WEST Pico Plus (Life Technologies SAS).
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2

Insulin-Stimulated Akt Signaling in Mice

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5 hour fasted mice were injected with intraperitoneal insulin (0.75 units/kg) and tissues harvested 15 min later were quickly frozen in liquid nitrogen and kept at −80°C until used for western blots as previously described (5 (link)). Antibodies used: anti-mouse CD36 (R&D Systems, AF1955), anti-pS473-Akt (Cell Signal Technology, 4060), anti-pT308-Akt (Cell Signal Technology, 13038) and total Akt (Cell Signal Technology, 4691).
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3

Detailed Antibody Panel for Signaling Assays

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The antibodies used in the study: mouse monoclonal anti-phosphotyrosine antibody (catalog 05–321, clone 4G10; Millipore); anti-pY1162/1163-IR-β (catalog 700393, clone 97H9L7; Invitrogen); anti-IR-β (catalog sc-711, clone C711; Santa Cruz Biotechnology Inc.); anti-actin (catalog A2228, clone AC-74, Sigma-Aldrich); anti-p-T308 AKT (catalog 13038, clone D25E6; Cell Signaling Tech), anti-AKT (catalog 4691, clone C67E7; Cell Signaling Tech), anti-JAK2 (catalog 3230, clone D2E12; Cell Signaling Tech); anti-catalase (catalog ab16731; abcam), anti-HA (catalog 11583816001, clone 12CA5; Roche), and anti-PTP1B (EP1841Y; abcam). Antibodies were used at dilutions recommended by the manufacturers.
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4

Western Blot Analysis of Protein Expression

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Cells were scraped from culture plates and incubated for 20 min in ice‐cold lysis buffer containing protease inhibitor cocktails. Nuclear and cytoplasmic fractions were prepared as described previously.[29 (link)
] Total protein (10 µg) was separated by SDS‐PAGE and transferred to a PVDF membrane (Bio‐Rad). The membrane was incubated with primary antibodies, and the protein was visualized with ECL (HRP) (Millipore). The following antibodies were used for western blot analysis: anti‐Pten (Cell Signaling Technology, 9188), anti‐β‐actin (Immunoway, YM3028), anti‐p‐T308‐Akt (Cell Signaling Technology, 13038), anti‐p‐S473‐Akt (Cell Signaling Technology, 4060), anti‐Akt (Cell Signaling Technology, 4691), anti‐cTnT (Abcam, ab8295), anti‐Dnmt3l (Cell Signaling Technology, 13451), anti‐Dnmt3b (Cell Signaling Technology, 48488), anti‐Dnmt3b (ab122932, Abcam), anti‐Igf1r (Cell Signaling Technology, 3027), anti‐p‐S253‐FoxO3a (Abcam, ab47285), anti‐p‐T32‐FoxO3a (Cell Signaling Technology, 9464), anti‐FoxO3a (Cell Signaling Technology, 2497), anti‐Phospho‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4370), anti‐ Insulin Receptor β (Abcam, ab69508), anti‐p44/42 MAPK (Erk1/2) (Cell Signaling Technology, 4695), anti‐β‐Tubulin (Cell Signaling Technology, 2146), and anti‐Lamin A/C (Cell Signaling Technology, 4777).
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5

Fas receptor signaling in EGFR-mediated cell growth

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The sources of antibodies and reagents are as followed: anti-pY291 Fas (prepared as previously described13 and validated for immunoblotting13 and flow cytometry Fig. S8); anti-Src, anti-Yes (R&D Systems); anti-human Fas (C20, for immunoblotting) and anti-Cyclin D1 (Santa Cruz); anti-Fas (Apo1–3, for co-immunoprecipitation) (Enzo); anti-EGFR, anti-pY1068 EGFR, anti-pY705 STAT3, anti-STAT3, anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pErk1/2, and anti-Histone H3 (Cell Signaling Technology); anti-Erk1/2 (Sigma); anti-GAPDH (Calbiochem); horseradish peroxidase conjugated anti-rabbit and anti-mouse (Jackson ImmunoResearch); anti-mouse IgG-Alexa Flour 488 (Life Technology); Recombinant human EGF (Gibco), recombinant amphiregulin (R&D Systems); protein G sepharose beads (Zymed Laboratories); WST-1 (Interchim); Matrigel (BD Bioscience). Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection according to the manufacturer’s protocol for reverse transfection.
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6

NMDAR and Signaling Pathway Antibodies

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The following primary antibodies were used in this study: anti-NR1 (#MAB1586 Millipore), anti-NR1C1 (#AB5046P Millipore), anti-NR2A (#ab14596 Abcam), anti-NR2B (#ab14400 Abcam), anti-NR1 C1 P-S897 (#ABN99 Millipore), anti-NR2B P-Y1472 (#AB5403 Millipore), anti-NR2B P-Y1303 (#ab81271 Abcam), anti-NR2B P-Y1480 (#PA1-4733 ThermoFisher Scientific), anti NR2B P-Y1252 (#ab18532 Abcam), anti-GSK3β (#9315 Cell Signaling Technology), anti-P-S9 GSK3β (#9336 Cell Signaling Technology), anti-P-Y216 GSK3β (#ab75745 Abcam), anti-Akt (#4691 Cell Signaling Technology), anti-P-S473 Akt (#4060 Cell Signaling Technology), anti-P-T308 Akt (#2965 Cell Signaling Technology), anti-PKC sampler kit (#611421 BD Biosciences), anti-PKCγ (#ab71558 Abcam), and anti-α tubulin (#T9026 Sigma Aldrich).
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7

Akt1/2 KO HCT116 Cell Line Transfection

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The Akt1/2 knockout HCT116 colon cancer cell line was given as a gift from Dr Bert Vogelstein (Johns Hopkins University; Ericson et al., 2010 (link)). Cells were cultured in McCoy’s 5A (Gibco) supplemented with 10% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco) at 37°C and 5% CO2. When the cell confluence reached around 70% in six-well plates, the medium was changed with McCoy’s 5A (Gibco) having 5% (v/v) FBS (Sigma) and 1% (v/v) penicillin/streptomycin (Gibco), and the cells were transfected with 1.5 μg of pcDNA3.1-Flag-HA-Akt plasmid or 1.5 μg (3.0 μg for Y18A mutant) of pcDNA3.1-Myr-HA-Akt plasmid complexed with 3 μL Lipofectamine 3000 (Invitrogen) and 3 μL P3000 reagent (Invitrogen) in Opti-MEM medium (Gibco) at 37°C and 5% CO2. Seventy-two hours after transfection, the cells were washed with cold PBS and lysed by adding 150 μL RIPA buffer (Cell Signaling Technology) containing ×1 complete protease inhibitor tablet (Thermo Fisher Scientific) and ×1 PhosStop tablet (Roche), and 1 mM PMSF for 10 min at 4°C. Thirty μg of total protein (BCA assay) was loaded on an SDS-PAGE gel. Membrane transfer and western blotting were carried out as described above with 1:1000 dilution for primary antibodies: anti-Akt, anti-pT308 Akt, anti-Foxo1, anti-Foxo3a, and anti-pT24 Foxo1/pT32 Foxo3a, anti-GAPDH (Cell Signaling Technology).
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