Anti -PRAS40, phosphorylated (Thr246) (Proline-Rich AKT Substrate 40kD Lobe, AKT151)
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| Clone |
Host |
Grade |
Applications |
| Polyclonal |
Rabbit |
Affinity Purified |
B |
|
| The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor binding protein that is phosphorylated directly by mTOR complex (mTORC) 1 but not mTORC 2 in vitro, predominantly at PRAS40 (Ser183). The binding of S6K1 and 4E-BP1 to raptor requires a “TOR signaling” (TOS) motif which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids; PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe129) to Ala; immediately carboxyterminal to Phe129 are two small hydrophobic amino acid followed by two acidic residues, an arrangement different from that of the canonical TOS motif. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site Ser183, to Asp. PRAS40 (Ser183) was phosphorylated in intact cells; as with S6K1 and 4E-BP1, PRAS40 (Ser183) phosphorylation was inhibited by rapamycin, by 2-deoxyglucose and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser183) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched-chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser183) and its binding to raptor. More importantly, RNAi-induced depletion of PRAS40 enhanced the amino-acid stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the capacity or ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo, and is therefore a potential locus of regulation, as suggested by others. | | | Catalog # | P5640-15A | | Applications | Suitable for use in Western Blot. Other applications not tested. | | Recommended Dilution | Western Blot: 1:1000 | | Optimal dilutions to be determined by the researcher. | | Storage and Stability | May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. | | Quality Control Testing Immunoblot Analysis | A 1:1000 dilution of this lot detected phosphorylated PRAS40 (Thr246) in RIPA lysates of unstimulated and PDGF stimulated NIH3T3 cells. Untreated (Lane 1) or PDGF treated (Lanes 2, 3, 4) lysates from NIH3T3 cells were resolved by electrophoresis, transferred to nitrocellulose, and probed with anti-phospho-PRAS40 (Thr246) for 2 hours at room temperature. Lane 3 was pre-incubated with the blocking peptide immunogen corresponding to phospho- PRAS40 (Thr246). Lane 4 blot was pre-incubated with phosphatase. Proteins were visualized via HRP and chemiluminescent detection. Arrow indicates phospho-PRAS40 (Thr246). | | Clone Type | Polyclonal | | Host | Rabbit | | Concentration | Not determined | | Form | Supplied as a liquid in PBS (without Mg2+ and Ca2+), pH 7.3 containing 1.0 mg/mL BSA (IgG, protease free) and 0.05% sodium azide and 50% glycerol. | | Purity | Purified by immunoaffinity chromatography. | | Immunogen | Peptide corresponding to amino acid region encompassing the human phospho-PRAS40 (Thr246). | | Specificity | Recognizes phosphorylated PRAS40 (Thr246)), Mr 40kDa. Species Cross-reactivity: Human and mouse tested. | | | Important Note | This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological. |
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