Total pras40

Manufactured by Cell Signaling Technology

Sourced in United States

Total PRAS40 is an ELISA kit designed to quantify the total amount of PRAS40 protein in cell and tissue lysates. PRAS40 is a regulatory subunit of the mTOR complex and plays a role in the control of cell growth and metabolism.

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6 protocols using total pras40

Leelamine Regulates Melanoma Cell Signaling

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Cell lysates were harvested by addition of RIPA lysis buffer and samples were processed as described. Briefly, 1.5×10⁶ melanoma cells were plated in 100 mm culture dishes and 48 hours later, treated with leelamine (3–6 µmol/L) for 3 to 24 hours. Protein lysates were collected for Western blotting and targets validation. Blots were probed with antibodies according to each supplier’s recommendations: antibodies to total Akt, phospho-Akt (Ser473), total PRAS40, phospho-PRAS40 (Thr246), total CREB, phospho-CREB (Ser133), phospho-p70 S6 kinase (Thr389), total Erk1/2, phospho-Erk1/2 (Thr202/Tyr 204), total Stat3, phospho-Stat3 (Tyr705) and cleaved PARP from Cell Signaling Technology (Danvers, MA); total PRAS40 from Invitrogen (Carlsbad, CA); cyclin D1, α-enolase and secondary antibodies conjugated with horseradish peroxidase from Santa Cruz Biotechnology (Santa Cruz, CA). Immunoblots were developed using the enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, Rockford, IL). Proteins or pathways not affected by leelamine treatment were listed in Supplementary Table 1.

Gowda R., Madhunapantula S.V., Kuzu O.F., Sharma A, & Robertson G.P. (2014). Targeting Multiple Key Signaling Pathways in Melanoma using Leelamine. Molecular cancer therapeutics, 13(7), 1679-1689.

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AKT Signaling Pathway Activation Analysis

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MCF10a or Ba/F3 cells stably expressing WT and mutant AKT were seeded in either 6-well plates or 60 mm dishes, and after overnight exposure to the assay medium, the cells were lysed, sonicated and 30 μg protein was loaded onto SDS-PAGE gels, transferred to nitrocellulose membranes, and immunoblotted for p-Akt and other downstream molecular targets of PI3K pathway activation. Antibodies for p-Akt (T308) (D25E6) [dilution 1:1000], p-Akt (S473) (D7F10 and D9E) [dilution 1:1000], p-PRAS40 (T246) [dilution 1:1000], p-S6RP (S240/244) [dilution 1:2000], p-GSK-3β (S9) [dilution 1:1000], and total PRAS40 [dilution 1:1000], S6RP [dilution 1:2500], GSK-3β [dilution 1:1000] were acquired from Cell Signaling Technology. V5 probe (E10) [dilution 1:2000] and β-actin antibodies (C4) [dilution 1:5000] were acquired from Santa Cruz Biotechnology.

Shrestha Bhattarai T., Shamu T., Gorelick A.N., Chang M.T., Chakravarty D., Gavrila E.I., Donoghue M.T., Gao J., Patel S., Gao S.P., Reynolds M.H., Phillips S.M., Soumerai T., Abida W., Hyman D.M., Schram A.M., Solit D.B., Smyth L.M, & Taylor B.S. (2022). AKT mutant allele-specific activation dictates pharmacologic sensitivities. Nature Communications, 13, 2111.

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Antibody Validation for Protein Analysis

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The following antibodies used in this study were purchased from Cell Signaling: acetyl-α-tubulin (1:1000; #5335), TAB1 (1:1000; #3226), pERK (1:2000; #9101), total ERK (1:2000; #4695), pAKT-S473 (1:1000; #4060), pP38 (1:2000; #4511), pJNK (1:2000; #9255), cMYC (1:2000; #13987), pGSK3β-Ser9 (1:2000; #5558), total GSK3β (1:2000; #12456), pTAK1 (1:1000; #9339), total TAK1 (1:1000; #4505), total AKT (1:1000; #4691), pPRAS40 (1:2000; #2997), total PRAS40 (1:2000; #2610), pSmad2 (1:1000; #8828), and Myc-Tag (1:4000; #2276). Total tubulin YOL1/34 (1:2000; MCA78G) (Abcam), αHA (1:3000; #11867431001), α-Flag (1:4000; F1804) (Sigma), αTAT1 phosphoserine 237 (1:250) (Thermo Scientific), and αTAT1 (1:500; sc-167354) (Santa Cruz) were also used.

Shah N., Kumar S., Zaman N., Pan C.C., Bloodworth J.C., Lei W., Streicher J.M., Hempel N., Mythreye K, & Lee N.Y. (2018). TAK1 activation of alpha-TAT1 and microtubule hyperacetylation control AKT signaling and cell growth. Nature Communications, 9, 1696.

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Immunoblotting Analysis of ROCK Signaling

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KPC or TKCC5 cells (5x10⁵) in complete media were plated into the wells of 6-well culture dishes and allowed to settle and grow overnight. Next day, cells were treated with inhibitors or DMSO vehicle in complete media for 1 h. Whole cell lysates were prepared with lysis buffer (1% SDS, 50 mM Tris pH 7.5), followed by protein concentration determination using the Bicinchoninic assay (Sigma). Standard protocols were used for immunoblotting (13 (link)). Membranes were incubated with primary antibodies: ROCK1 (BD-611136), ROCK2 (BD-610623), ROCK1/2 (Millipore 07-1458), total AKT (Cell Signaling 2920S), phospho-AKT (Cell Signaling 4060S), phospho-MYPT1 (Millipore 36003), phospho-MLC2 (Cell Signaling 3674), total MLC (MRCL3/MRLC2/MYL9; Santa Cruz sc-28329), phospho-PRAS40 (Cell Signaling 2997S), total PRAS40 (Cell Signaling 2691S), GAPDH (Millipore MAB374) and secondary antibodies: Alexa-Fluor 680 and DyLight 800 (Thermo Fisher Scientific)-conjugated antibodies. Infrared imaging (Li-Cor Odyssey) was used for detection and quantification of protein signals.

Rath N., Munro J., Cutiongco M.F., Jagiełło A., Gadegaard N., McGarry L., Unbekandt M., Michalopoulou E., Kamphorst J.J., Sumpton D., Mackay G., Vennin C., Pajic M., Timpson P, & Olson M.F. (2018). Rho kinase inhibitor AT13148 blocks pancreatic ductal adenocarinoma invasion and tumor growth. Cancer research, 78(12), 3321-3336.

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Intracellular Redox Signaling Pathways

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Antibodies purchased from Cell Signaling Technology were to phospho-IGF receptor (Tyr1135), totalIGF receptor, phospho-Akt (Ser473), total-Akt, phospho-PRAS40 (Thr246), total PRAS40, phospho-p38 (Thr180/Tyr182), total-p38, phospho-ERK (Thr202/Tyr204), total-ERK, phospho-JNK (Thr183/Tyr185), total JNK, phospho c-Jun (ser73), total c-Jun, phospho-Prx1 (Tyr194), total-Prx1 and β-tubulin. Antibodies purchased from Abcam were to PrxSO_2/3, Prx2, Prx3 and β-actin. The Orp1-roGFP cellular hydrogen peroxide sensor (Premo™) was purchased from Life Technologies. Menadione, DMNQ, Nethylmaleimide (NEM) and catalase were purchased from Sigma Aldrich.

Collins J.A., Wood S.T., Bolduc J.A., Nurmalasari N.P., Chubinskaya S., Poole L.B., Furdui C.M., Nelson K.J, & Loeser R.F. (2019). Differential Peroxiredoxin Hyperoxidation Regulates MAP Kinase Signaling in Human Articular Chondrocytes. Free radical biology & medicine, 134, 139-152.

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Western Blot Analysis of Cellular Proteins

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Cells were lysed in cell lysis buffer (20 mM Tris, pH 7.5, 135 mM NaCl, 5% [v/v] glycerol, 50 mM NaF, 0.1% [v/v] Triton X-100, plus protease inhibitors), centrifuged and protein quantified using Bradford reagent (Sigma-Aldrich). Samples were made up in NuPAGE LDS sample buffer (Life Technologies). Western blotting was performed as previously described [28 (link)]. Blots shown are representative of 3 independent experiments. Anti-β-actin (#4967), phospho-TSC2 S1387 (#5584), total TSC2 (#3990), IRE1α (3294S), phospho-S6K1 T389 (#9205), total S6K1 (#9202), phospho-rpS6 S235/236 (#2211), total rpS6 (#2217), phospho-4E-BP1 S65 (#9451), total 4E-BP1 (#9644), phospho-ACC S79 (#3661), total ACC (#3676), PARP (#9542), caspase 3 (#9662), ATF4 (#11815), phospho-FOXO3a (#9466S), total FOXO3a (#9467), phospho-PRAS40 T246 (#2997), total PRAS40 (#2691) and phospho-Bad S136 (#4366) antibodies were from Cell Signaling Technology (Danvers, MA, USA). GADD34 antibody (10449-1-AP) was from Proteintech (Manchester, UK).

Dunlop E.A., Johnson C.E., Wiltshire M., Errington R.J, & Tee A.R. (2017). Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells. Oncotarget, 8(30), 48711-48724.

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