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Rabbit anti phosphorylated akt

Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom

Rabbit anti-phosphorylated Akt is a primary antibody that specifically recognizes the phosphorylated form of the Akt protein. Akt is a serine/threonine protein kinase that plays a central role in the regulation of cell growth, proliferation, and survival.

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5 protocols using rabbit anti phosphorylated akt

1

Protein Analysis of Wound Tissue

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Total protein was isolated from the wound tissue obtained at indicated times with RIPA (Aspen Inc.) following the manufacturer’s instruction. The protein concentrations were determined using the BCA (Aspen Inc.). Equal amounts of protein from each tissue were separated by SDS-PAGE (Aspen Inc.), transferred to a PVDF membrane, and incubated with 5% milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with rabbit anti-phosphorylated-ERK (1:1,000; Cell Signaling Technology), rabbit anti-ERK (1:2,000; Cell Signaling Technology), rabbit anti-phosphorylated-AKT (1:1,000; Cell Signaling Technology), rabbit anti-AKT (2:1,000; Cell Signaling Technology), rabbit anti-SPRED1 (1:1,000; ABCAm), mouse anti-VEGF (1:500; ABCAm), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1:1,000; ABCAm). Next, anti-rabbit IgG (1:10,000; Aspen, Inc.) and anti-mouse IgG (1:10,000; Aspen, Inc.) were used as second antibodies at 1:2,000 for 30 min. Quantification of the protein bands were performed with AlphaEaseFC (Alpha Innotech, San Leandro, CA) software. Sample analyses were performed on day 9.
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2

Western Blot Analysis of Cellular Proteins

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Protein extracts were subjected to 8%–12% SDS-PAGE and transferred to the PVDF membrane (Millipore, Hayward, CA, USA). Rabbit anti-NARFL (1:1000, Abclonal, Wuhan, China), rabbit anti-HIF-1α (1:500, Boster, Wuhan, China), rabbit anti-Akt (1:1000, Cell Signaling Technology, MA, USA), rabbit anti-phosphorylated Akt (1:1000, Cell Signaling Technology, MA, USA), mouse anti-β-Actin (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as the primary antibodies. The secondary antibodies conjugated to horseradish peroxidase were used at 1:5000 dilutions (proteintech, Wuhan, China).
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3

Immunofluorescence Staining of Brain Sections

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For immunofluorescence experiments, 40–50 μm floating sections were collected in 1× PBS, then rinsed once in 1× PBT (PBS + 1% Triton 100×), and incubated overnight at 4 °C in a cocktail of primary antibodies that were diluted in 1× PBT + 20% normal donkey serum. After the overnight incubation, sections were rinsed three times with 1× PBT for 10 min and incubated for 1.5 h in the corresponding secondary antibodies (1:500, Jackson-ImmunoResearch or Invitrogen). Cerebellar sections were then washed three times with 1× PBT for 10–15 min, incubated with DAPI, and mounted in Poly-aquamount (Polysciences). The following primary antibodies were used: rabbit anti-IBA1 (1:200, Wako, # 019-19741), mouse anti-CALB (calbindin, 1:200, Sigma-Aldrich, #C9848), rat anti-CD68 (1:200, Bio-Rad, #MCA1957), Lycopersicon esculentum(Tomato)-Lectin (1:200, Sigma-Aldrich, #L0401), guinea-pig anti-VGLUT1 (1:800, Synaptic Systems, #135404), rabbit anti-phosphorylated S6R (1:200, Cell Signaling, #2211), rabbit anti-phosphorylated AKT (1:200, Cell Signaling, #8112), rabbit anti-TFEB (1:200, Bethyl Laboratories, A303-673A), rabbit anti-FASN (1:200, Cell Signaling, #3180), rabbit anti-pyruvate dehydrogenase E 1 alpha (PDE) (1:200, GeneTex, # GTX104015), rat anti-CD107a (LAMP-1) (1:200, BioLegend, # 121602).
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4

Immunoblot Analysis of Phosphorylated Akt and p38

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After 120 min stimulation of B cells isolated from healthy donors by anti-IgG/IgM antibody with or without pre-IFN treatment, cells were lysed with 1x SDS sample buffer (Cell Signaling Technologies) and sonicated. The cell lysates were electrophoresed on 12% polyacrylamide gel and then transferred onto Immobilon transfer membranes (Millipore). The membranes were treated with rabbit anti-phosphorylated Akt (Cell Signaling Technologies), rabbit anti-phoshorylated p38 (Cell Signaling Technologies), and rabbit anti-tubulin antibody (abcam, Cambridge, UK). IRDye 800CW-conjugated goat anti-rabbit (LI-COR Biosciences, Lincoln, NE) was used as a secondary antibody. Fluorescence intensity was measured, and densitometric analysis was performed with an Odyssey Infrared Imaging System (LI-COR Biosciences).
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5

Immunoblotting Analysis of Protein Signaling

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Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and a phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galecint‐1 (1:1000, R&D systems), mouse anti‐TSC22D3 (1:500), mouse anti‐ubiquitin (1:500, Santa Cruz Biotechnology), rabbit anti‐HIF‐1α (1:1000), rabbit anti‐phosphorylated AKT (1:2000), rabbit anti‐AKT (1:2000), rabbit anti‐phosphorylated ERK1/2 (1:2000), rabbit anti‐ERK1/2 (1:2000, Cell signaling technology) and rabbit anti‐β‐actin (1:4000, Medical & Biological Laboratories) antibodies. Horseradish peroxidase‐conjugated anti‐goat, anti‐mouse and anti‐rabbit IgGs (Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were visualized using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
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