Human phospho-PRAS40 (T246) Antibody (1211A)

R&D Systems | Catalog # MAB68901

Recombinant Monoclonal Antibody.
R&D Systems

Key Product Details

Validated by

Biological Validation

Species Reactivity

Human

Applications

Western Blot

Label

Unconjugated

Antibody Source

Recombinant Monoclonal Rabbit IgG Clone # 1211A
View all PRAS40 Primary Antibodies »

Product Specifications

Immunogen

Phosphopeptide containing the human PRAS40 T246 site
Accession # Q96B36

Specificity

Detects human and mouse PRAS40 when phosphorylated at T246.

Clonality

Monoclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for Human phospho-PRAS40 (T246) Antibody (1211A)

Detection of Human and Mouse Phospho-PRAS40 (T246) antibody by Western Blot.

Detection of Human and Mouse Phospho-PRAS40 (T246) by Western Blot.

Western blot shows lysates of MCF-7 human breast cancer cell line and NIH-3T3 mouse embryonic fibroblast cell line untreated (-) or treated (+) with 100 ng/ml Recombinant Human IGF-I (Catalog # 291-G1) for 60 minutes and 100 ng/ml Recombinant Human PDGF-BB (Catalog # 220-BB) for 20 minutes. PVDF membrane was probed with 1 µg/mL of Rabbit Anti-Human Phospho-PRAS40 (T246) Monoclonal Antibody (Catalog # MAB68901) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-PRAS40 (T246) at approximately 40 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Detection of Phospho-PRAS40 (T246) by Western Blot

Detection of Phospho-PRAS40 (T246) by Western Blot

The combination of high glucose and oxLDL promotes sCD14 shedding though Akt-PRAS40 pathway activation. MDM were treated with 150 μg/mL of oxLDL for 24 h in culture medium containing 5 mM (normal glucose, NG) or 15 mM (high glucose, HG) of glucose. (A) MDM lysates were exposed to phospho-kinase array spotted membranes. Upper panel: dot images. Lower graph: quantification of dot intensities. (B) Cell lysates were probed with anti-pPRAS40 (Thr246) by Western blotting, and equal loading was determined by probing with alpha -tubulin. Upper panel: representative Western blot images, lower panel: mean ± SEM fold induction levels relative to untreated MDMs (NG) from 2 independent donors. (C–F) Cells were incubated for 45 min with 10 µM of wortmannin (W) for 24 h prior to oxLDL stimulation under NG or HG conditions. (C) PRAS40 (Thr246) and Akt (Ser473) phosphorylation determined by Western blotting. Upper panel: representative images. Lower panel: graph showing fold induction levels relative to untreated cells (NG) from 4 donors. (D) TLR4 and CD14 mRNA expression analyzed by RT-PCR. The data show the mean fold change relative to untreated cells (NG) ± SEM from 4 donors. (E) sCD14 in culture supernatants measured by Western blotting. Upper panel: representative Western blot images. Lower panel: band intensity values/total protein. Fold induction levels relative to untreated cells (NG) from 4 donors are shown. (F) THP1 MΦ were pre-incubated with wortmannin (W) and then treated with NG, HG + oxLDL, and with 2 µg/mL of rsCD14. After 24 h, TNF in culture supernatants was measured by ELISA. Graph showing the mean ± SEM from 3 independent experiments performed in triplicate (* p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney test). MDM: monocyte-derived macrophages. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37237950), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Phospho-PRAS40 (T246) by Western Blot

Detection of Phospho-PRAS40 (T246) by Western Blot

The combination of high glucose and oxLDL promotes sCD14 shedding though Akt-PRAS40 pathway activation. MDM were treated with 150 μg/mL of oxLDL for 24 h in culture medium containing 5 mM (normal glucose, NG) or 15 mM (high glucose, HG) of glucose. (A) MDM lysates were exposed to phospho-kinase array spotted membranes. Upper panel: dot images. Lower graph: quantification of dot intensities. (B) Cell lysates were probed with anti-pPRAS40 (Thr246) by Western blotting, and equal loading was determined by probing with alpha -tubulin. Upper panel: representative Western blot images, lower panel: mean ± SEM fold induction levels relative to untreated MDMs (NG) from 2 independent donors. (C–F) Cells were incubated for 45 min with 10 µM of wortmannin (W) for 24 h prior to oxLDL stimulation under NG or HG conditions. (C) PRAS40 (Thr246) and Akt (Ser473) phosphorylation determined by Western blotting. Upper panel: representative images. Lower panel: graph showing fold induction levels relative to untreated cells (NG) from 4 donors. (D) TLR4 and CD14 mRNA expression analyzed by RT-PCR. The data show the mean fold change relative to untreated cells (NG) ± SEM from 4 donors. (E) sCD14 in culture supernatants measured by Western blotting. Upper panel: representative Western blot images. Lower panel: band intensity values/total protein. Fold induction levels relative to untreated cells (NG) from 4 donors are shown. (F) THP1 MΦ were pre-incubated with wortmannin (W) and then treated with NG, HG + oxLDL, and with 2 µg/mL of rsCD14. After 24 h, TNF in culture supernatants was measured by ELISA. Graph showing the mean ± SEM from 3 independent experiments performed in triplicate (* p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney test). MDM: monocyte-derived macrophages. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/37237950), licensed under a CC-BY license. Not internally tested by R&D Systems.

Applications for Human phospho-PRAS40 (T246) Antibody (1211A)

Application
Recommended Usage

Western Blot

1 µg/mL
Sample: MCF‑7 human breast cancer cell line and NIH‑3T3 mouse embryonic fibroblast cell line treated with Recombinant Human IGF‑I (Catalog # 291-G1) and Recombinant Human PDGF‑BB (Catalog # 220-BB)

Formulation, Preparation, and Storage

Purification

Protein A or G purified from cell culture supernatant

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS. For liquid material, refer to CoA for concentration.

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 12 months from date of receipt, -20 to -70 °C as supplied.
  • 1 month, 2 to 8 °C under sterile conditions after reconstitution.
  • 6 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: PRAS40

PRAS40 (Proline-rich AKT1 substrate 1), also known as Akt1S1 and p39, is a 40-42 kDa cytoplasmic phosphoprotein that lacks generally recognized structural motifs. It is widely expressed and is considered to be key regulator of mTORC1 (mTOR plus Raptor and G beta L), a complex through which Akt signals into the cell. Through phosphorylation, mTORC1 activity is upregulated by PRAS40. In particular, nonphosphorylated PRAS40 binds to and serves as a negative regulator of mTORC1 activity. Upon Insulin signaling, PRAS40 is phosphorylated on Thr246, Ser221 and Ser183. This causes it to bind 14-3-3 and results in its dissociation from mTORC1, freeing up mTOR to regulate (positively or negatively) protein synthesis. Human PRAS40 is 256 amino acids (aa) in length. It contains one extended Pro-rich region (aa 35-96) plus at least nine utilized Ser/Thr phosphorylation sites. There is one alternative start site at Met131. Over aa 119-256, human PRAS40 shares 93% aa sequence identity with mouse PRAS40.

Long Name

40 kDa Proline-rich Akt1 Substrate

Alternate Names

AKT1S1, Lobe

Entrez Gene IDs

84335 (Human); 67605 (Mouse); 292887 (Rat)

Gene Symbol

AKT1S1

UniProt

Q96B36 (Human)

Additional PRAS40 Products

Product Documents for Human phospho-PRAS40 (T246) Antibody (1211A)

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Product Specific Notices for Human phospho-PRAS40 (T246) Antibody (1211A)

For research use only

Related Research Areas

Autophagy
Interferons

Citations for Human phospho-PRAS40 (T246) Antibody (1211A)

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