COS-7 cells were seeded on to 22 mm poly-D-lysine-treated glass coverslips in a 6-well plate, grown for approximately 20 h, serum starved for 4 h, and treated with or without 100 nM insulin for 10 min. Cells were rinsed briefly in PBS and fixed with 4% paraformaldehyde for 15 min at room temperature followed by incubation with 100%methanol for 3 min at −20°C. Cells were rinsed again in PBS three times followed by permeabilization and blocking in PBS containing 0.25% Triton X-100, 5% BSA and 5% goat serum for 15 min at room temperature. Cells were incubated in a humidified chamber for 1 h at room temperature with either a PKCζ-specific antibody (Santa Cruz Biotechnology sc-216 diluted 1:40, also detects PKCλ) or a pan-Akt antibody (Cell Signaling 9272, diluted 1:200). Cells were then rinsed three times in PBST and incubated in a humidified chamber protected from light for 30 min at room temperature with an AlexaFluor 488-conjugated secondary antibody (Invitrogen A110034, diluted 1:500). Cells were rinsed a final three times in PBST prior to mounting the coverslips on glass slides and imaging via a 40× objective on a Zeiss Axiovert fluorescent microscope (Carl Zeiss Microimaging) using a MicroMax digital camera (Roper-Princeton Instruments) controlled by MetaFluor software version 3.0 (Universal Imaging).
Pan akt antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Pan-Akt antibody is a laboratory reagent used for the detection and analysis of Akt proteins, also known as protein kinase B (PKB). It recognizes all three isoforms of Akt (Akt1, Akt2, and Akt3) and can be used in various immunoassay techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and activation of Akt in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt ser473 antibody, by Cell Signaling Technology (2 mentions)
Axiovert fluorescent microscope, by Zeiss (1 mentions)
Anti p akt ser473 antibody, by Cell Signaling Technology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Mtor antibody, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc pi apoptosis kit, by Merck Group (1 mentions)
Phospho akt thr308 antibody, by Cell Signaling Technology (1 mentions)
Phospho mtor ser2448 antibody, by Cell Signaling Technology (1 mentions)
Cck 8 kit, by MedChemExpress (1 mentions)
St7l antibody, by Santa Cruz Biotechnology (1 mentions)
Bs 0295g cy3, by Bioss Antibodies (1 mentions)
Dmi4000b microscope, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail 2 and 3, by Merck Group (1 mentions)
6 protocols using pan akt antibody
1
Insulin-Stimulated PKCζ and Akt Localization in COS-7 Cells
2
Western Blot Protein Analysis Protocol
3
Quantitative Akt-CTMP Interaction in ALS
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Co-immunoprecipitation of Akt total protein and bound CTMP from mSOD1G93A gastrocnemius muscle followed previously published protocols with modification (Wu et al., 2017). In brief, Bio-Rad Rapid Magnetic Beads (Bio-Rad) were used to bind pan-Akt antibody (Cell Signaling, 1:50) and this complex was allowed to immunoreact with PD35 and end-stage mSOD1G93A mouse gastrocnemius muscle protein lysate under rotation for 1 hr at room temperature. Non-antibody protein exposure (beads only) was used as a negative control. The bound protein was eluted for Western blotting via addition of 1x Laemmli sample buffer (Bio-Rad) and heating at 70 °C for 10 min. After heating, the sample buffer containing the immunoprecipitated and associated proteins was transferred to a separate tube for Western blot.
4
Apoptosis Assay with Akt/mTOR Signaling
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The CCK8 kit was purchased from MedChemExpress in the United States, the transwell cell was purchased from Corning in the United States, and the Annexin V-FITC/PI apoptosis kit was purchased from Sigma-Aldrich in the United States. Phospho-Akt (Ser473) antibody, Phospho-Akt (Thr308) antibody, Akt (pan) antibody, mTOR antibody, and Phospho-mTOR (Ser2448) antibody were purchased from Cell Signaling Technology, USA.
5
Immunofluorescence Analysis of AKT and ST7L
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Cells cultured on glass coverslips were fixed with 4% paraformaldehyde, blocked with 5% bovine serum albumin and incubated with AKT (pan) antibody (no.4691; Cell Signaling Technology) and ST7L antibody (sc-138649; Santa Cruz, Biotechnology) overnight at 4 °C. Then, cells were incubated with mouse anti-goat IgG/FITC antibody (bs-0294M-FITC, BIOSS) and goat anti-rabbit IgG/Cy3 antibody (bs-0295G-Cy3, BIOSS) for 2 h at room temperature. DAPI (Invitrogen) was used to stain the nucleus. LEICA DMI4000B microscope (Leica, Heidelberg, Germany) was used to take the images.
6
Insulin-Stimulated Akt Signaling Pathway
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