Pras40

Manufactured by Cell Signaling Technology

Sourced in United States

PRAS40 is a protein that acts as a regulatory subunit of the mTOR complex 1 (mTORC1). It plays a role in modulating the activity of mTORC1, which is involved in various cellular processes such as cell growth, proliferation, and metabolism.

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Lab products found in correlation

Close spelling variants of this product

P-PRAS40 (25 citations) Phospho-PRAS40 (19 citations) P-PRAS40 (T246) (13 citations) P-PRAS40 Thr246 (10 citations) Anti-PRAS40 (10 citations) Total PRAS40 (6 citations) Anti-p-PRAS40 (Thr246) (5 citations) Anti-phospho-PRAS40 (T246) (4 citations) Phospho-PRAS40 (T246) (3 citations) Anti-phospho-PRAS40 (2 citations)

44 protocols using pras40

Protein Synthesis Signaling Pathway Analysis

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Protein content was compared across groups via Western Blot analysis using previously described methods [12 (link), 16 (link)]. 4-hydroxynonenal (4-HNE) conjugated proteins were measured as an index of oxidative damage (Abcam, Cambridge, MA; product #46546). To measure markers of protein synthesis signaling, membranes were incubated with antibodies against phosphorylated Akt (Ser 473), phosphorylated proline rich Akt substrate of 40kD (PRAS40) (Thr 246), phosphorylated mammalian target of rapamycin (mTOR) (Ser 2448), and phosphorylated 4E-binding protein 1 (4E-BP1) (Thr 37/46), all purchased from Cell Signaling Technology (Danvers, MA). Subsequently, membranes were stripped, blocked, and incubated with antibodies against total Akt, GSK3β, PRAS40, mTOR, ribosomal protein s6, and 4E-BP1 all purchased from Cell Signaling Technologies (Danvers, MA). For all western blots where both phosphorylated protein and total protein were measured, the data were analyzed as a ratio of the phosphorylated to total protein ratio by dividing the density of phosphorylated protein by the density of total protein. All western blots were normalized to α-tubulin as a loading control. Product numbers for specific antibodies from Cell Signaling Technologies are as follows: Akt (#2920), pAkt (#4058), PRAS40 (#2691), PRAS40 (#2997), p-mTOR (#5536), mTOR (#2983), 4E-BP1 (#9452), and p-4E-BP1 (#9459).

Hudson M.B., Smuder A.J., Nelson W.B., Wiggs M.P., Shimkus K.L., Fluckey J.D., Szeto H.H, & Powers S.K. (2015). Partial Support Ventilation and Mitochondrial-Targeted Antioxidants Protect against Ventilator-Induced Decreases in Diaphragm Muscle Protein Synthesis. PLoS ONE, 10(9), e0137693.

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Molecular Signaling Pathways in Neurological Research

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Primary antibodies were purchased as follows: P-PRAS40 (Thr246; cat. no. 2997; Cell Signaling Technology, Inc.), PRAS40 (cat. no. 2691; Cell Signaling Technology, Inc.), p-mTOR (Ser2448; cat. no. 5536; Cell Signaling Technology, Inc.), mTOR (cat. no. 2983; Cell Signaling Technology, Inc.), p-AKT (Ser473; cat. no. 4060; Cell Signaling Technology, Inc.), AKT (cat. no. 4685; Cell Signaling Technology, Inc.), p-P70S6K (Thr389; cat. no. 9234; Cell Signaling Technology, Inc.), P70S6K (cat. no. 14130; Cell Signaling Technology, Inc.), Light chain 3-I/II (LC3-I/II; cat. no. 12741; Cell Signaling Technology, Inc.), 14-3-3 (cat. no. 8312; Cell Signaling Technology, Inc.), P62 (cat. no. 18420-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. sc-365062; 1:2,000; Santa Cruz Biotechnology, Inc.). Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. sc-2054; Santa Cruz Biotechnology, Inc.). The following drugs were obtained from commercial sources as follows: Pilocarpine (cat. no. S4231; Selleck Chemicals), lithium chloride (cat. no. L9650; Sigma-Aldrich; Merck KGaA), Scopolamine (cat. no. S2508; Selleck Chemicals) and LY3023414 (cat. no. S8322; Selleck Chemicals).

Lin J., Fang Y., Zhang M., Wang X., Li L., He M., Xue A., Zhu K., Shen Y, & Li B. (2020). Phosphorylation of PRAS40 contributes to the activation of the PI3K/AKT/mTOR signaling pathway and the inhibition of autophagy following status epilepticus in rats. Experimental and Therapeutic Medicine, 20(4), 3625-3632.

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Western Blot Analysis of AKT/mTOR Pathway

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Western blots were performed as described previously [52 (link)]. Briefly, cells were lysed in RIPA buffer supplemented with phosphatase (Sigma–Aldrich) and protease inhibitors (Sigma–Aldrich) and equal amounts of protein were loaded and separated by SDS-PAGE and transferred onto a PVDF membrane. Membranes were probed with primary antibody then with appropriate horseradish peroxidase-conjugated secondary and incubated with ECL. Antibodies obtained from Cell Signalling: AKT-T (#9272), pAKT-Ser473 (#4060), pAKT-Thr308 (2965), S6 (#2217), pS6-Ser235/236 (#2111), PRAS40 (#2691), pPRAS40-Thr246 (#13175), TSC2 (#4308), PARP (#9532, #9542), MCL1 (#9429), BAD-T (#9239), BAD-P Ser 136 (#4366), Bcl-XL (#2764), Bcl-w (#2724), BAX (#5023, #2772), BAK (#12105, #6947), Bim (#2933), BID (#2002), Puma (#4976), SHIP2 (#2839). Other antibodies used were Bcl-2 (ab32124, Abcam), and HRK (#ab45419, Abcam) Actin (A2228, Sigma), Vinculin (#V9132, Sigma) PIK3R2 (#A302-593A, Bethyl Laboratories), Caspase-3 (#14220). Secondary antibodies used were HRP-linked anti-rabbit IgG (CST#7074,1:2000) and HRP-linked anti-mouse IgG (CST#7076, 1:2000).

Dunn S., Eberlein C., Yu J., Gris-Oliver A., Ong S.H., Yelland U., Cureton N., Staniszewska A., McEwen R., Fox M., Pilling J., Hopcroft P., Coker E.A., Jaaks P., Garnett M.J., Isherwood B., Serra V., Davies B.R., Barry S.T., Lynch J.T, & Yusa K. (2022). AKT-mTORC1 reactivation is the dominant resistance driver for PI3Kβ/AKT inhibitors in PTEN-null breast cancer and can be overcome by combining with Mcl-1 inhibitors. Oncogene, 41(46), 5046-5060.

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Immunoblot Analysis of Signaling Pathways

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Protein samples were resolved by SDS-PAGE and transferred to PVDF membranes prior to incubation at 4°C with indicated primary antibodies, mTOR and pMTOR^Ser2448, pAKT^Thr308 and AKT, Phospho-p44/42 MAPK (Erk1/2)^{Thr202/Tyr204}, p44/42 MAPK (Erk1/2) pPRAS40^Thr246, pSTAT3^Tyr705, STAT3, PRAS40 and β-Actin antibodies were purchased from Cell Signaling Technology, pHck^Tyr410 (Thermo Fisher Scientific) MICA/B (R&D Systems, Minneapolis, MN) and HSF-1 and HSF-1^Ser326 (abcam). Western blotting images chosen as representative depictions in the figures demonstrate equivalent results taken from biological replicates (N≥3).

Smalley M., Natarajan S.K., Mondal J., Best D., Goldman D., Shanthappa B., Pellowe M., Dash C., saha T., Khiste S., Ramadurai N., Eton E.O., Smalley J.L., Brown A., Thayakumar A., Rahman M., Arai K., Kohandel M., Sengupta S, & Goldman A. (2020). Nano-Engineered Disruption of Heat shock protein 90 (Hsp90) Targets Drug-Induced Resistance and Relieves Natural Killer Cell Suppression in Breast Cancer. Cancer research, 80(23), 5355-5366.

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Immunoblot Analysis of AKT Pathway

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All antibodies were purchased from Cell Signaling Technology: p-AKT Ser⁴⁷³ (#4060, 1:1000), p-AKT Thr³⁰⁸ (#2965, 1:1000), AKT (#4691, 1:1000), p-PRAS40 Thr²⁴⁶ (#2997, 1:1000), PRAS40 (#2691, 1:1000), PARP (#9542, 1:1000), p-S6 Ser^240/244 (#2215, 1:1000), p-RxxS/T (#9614, 1:1000); HA-tag (#2367, 1:1000); xCT/SLC7A11 (#12691, 1:1000); β-actin (#4970, 1:5000); conformation specific mouse anti-rabbit IgG HRP conjugate (#5127, 1:3000).

Lien E.C., Ghisolfi L., Geck R.C., Asara J.M, & Toker A. (2017). Oncogenic PI3K Promotes Methionine Dependency in Breast Cancer Cells Through the Cystine-Glutamate Antiporter xCT. Science signaling, 10(510), eaao6604.

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Isolation and Analysis of Cellular Fractions

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Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described^{21 (link)}. The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×^{21 (link)}), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).

Yi W., Gupta S., Ricker E., Manni M., Jessberger R., Chinenov Y., Molina H, & Pernis A.B. (2017). The mTORC1-4E-BP-eIF4E axis controls de novo Bcl6 protein synthesis in T cells and systemic autoimmunity. Nature Communications, 8, 254.

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Western Blot Analysis of Protein Signaling

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Tissue was homogenized, or cultured cells washed, and then lysed in complete RIPA buffer as described previously [50 (link)]. Primary antibodies (Ab) used: AR (sc-816), PI3K p110β (sc-376641), β-actin (sc-47778), GAPDH (sc-32233), and α-tubulin (sc-5286) from Santa Cruz Biotechnology (Santa Cruz, CA); AKT1 (#2938), AKT2 (#3063), AKT3 (#8018), AKT3 (#14982), AKT^poS473 (#9018), AKT^poS473/4 (#9271), AKT2^poS474 (#8599), AKT^poT308 (#4056S), phospho-AKT substrate RXXS*/T* (#9614S), PRAS40 (#2691S), PRAS40^poT346 (#2997T), PTEN (#9552), and PI3K p110α (#4249) from Cell Signaling Technologies (Beverly, MA); SMAD4 (ab-40759) from Abcam (Cambridge, MA). Between 15 and 35 μg of total protein per sample was separated by SDS-PAGE for IB.

Miller K., Degan S., Wang Y., Cohen J., Ku S.Y., Goodrich D, & Gelman I. (2023). PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression. Research Square.

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Immunoblotting for Signal Pathway Proteins

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Tumors and cells were lysed in RIPA lysis buffer with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Equal amount of total protein lysates were used for immunoblotting. The following antibodies were from Cell Signaling Technology (Beverly, MA) and were used at 1:1000 dilution: pAKT (#3787), AKT (#4691), pERK (#4370), ERK (#4695), pMET (#3077), MET (#3127), pEGFR (#3777), EGFR (#4267), p PRAS40 (#2997), PRAS40 (#2691), Vinculin (#13901). Other antibodies used in this paper are as follows: INPP4B (Abcam, Ab81269), beta-actin (Sigma, A2228).

Liu H., Paddock M.N., Wang H., Murphy C.J., Geck R.C., Navarro A.J., Wulf G.M., Elemento O., Haucke V., Cantley L.C, & Toker A. (2020). The INPP4B Tumor Suppressor Modulates EGFR Trafficking and Promotes Triple Negative Breast Cancer. Cancer discovery, 10(8), 1226-1239.

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Western Blot Analysis of Key Signaling Proteins

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The total cells were treated in lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.2), 5 mM EDTA, 0.1% Triton X-100, 5% glycerol, and 2% SDS]. Each amount of the protein extracts were denatured by boiling at 95 °C for 10 min in sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The total proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking at room temperature for 1 h using PBST containing 10% BSA with gentle shaking, the membranes were incubated with primary antibodies (1:1000) overnight at 4 °C with gentle shaking, incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.), and then visualized with the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ, USA). All of the primary antibodies (mTOR, PDK1, PRAS40, 4EBP-1, P70S6K, S6, Raptor, Rictor, eIF4E, eIF4G, HIF-1α, Cyclin D1, C-myc, Bcl2, VEGF, PARP, Caspase-3, Tubulin, GAPDH and β-actin) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Liu J., Liu Y., Zhang J., Liu D., Bao Y., Chen T., Tang T., Lin J., Luo Y., Jin Y, & Zhang J. (2020). Indole hydrazide compound ZJQ-24 inhibits angiogenesis and induces apoptosis cell death through abrogation of AKT/mTOR pathway in hepatocellular carcinoma. Cell Death & Disease, 11(10), 926.

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Western Blot Analysis of AKT Signaling

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Western analyses were performed as described25 (link). For each experiment, lysates from a cell line grown with or without serum were run on the same gel and all gels were run at the same time. Two gels were transferred to a single membrane. If more than one membrane was used for an experiment, a single solution for each primary and secondary antibody pair was made and divided between the two membranes that were subsequently hybridized in separate trays. Antibodies used included pAKT(S473) (product #4060), pAKT(T308) (product #2965); pan-AKT (product #2920 and 4691), p PRAS40 (product #2997), PRAS40 (product # 2610), pS6(S235) (product #2211), pS6(S240) (product #5364) S6 (product #2317 and 2217), pFOXO1/FOXO3a (product #9464), FOXO3a (product #2497), p GSK3α/β (product #9331), GSK3α/β (product #5676), pBAD (product #4366) and BAD (product #9239) from Cell Signaling Technologies (Danvers, MA, USA) or β-actin (product #A5441) from Sigma-Aldrich (St. Louis, MO, USA). Membranes were scanned and intensities calculated using a semi-automated infrared imaging system (Odyssey scanner and Image Studio Lite software, LiCor BioSciences Inc., Lincoln, NE, USA). Histograms show ratios of infrared signal intensities of indicated antibodies. For experiments using two membranes, histograms of infrared signal ratios graphed by membrane are shown in Supplementary Fig. 8.

Lindhurst M.J., Yourick M.R., Yu Y., Savage R.E., Ferrari D, & Biesecker L.G. (2015). Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome. Scientific Reports, 5, 17162.

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