Total s6k1

Manufactured by Cell Signaling Technology

Sourced in United States

Total S6K1 is a lab equipment product used to quantify the total amount of S6K1 protein in a sample. S6K1 is a serine/threonine-protein kinase that plays a key role in the regulation of cell growth, proliferation, and protein synthesis. This product provides a reliable and accurate method for measuring the total S6K1 levels in various biological samples.

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Lab products found in correlation

11 protocols using total s6k1

Nuclear Protein Extraction and Immunoblotting

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Cells were lysed in NP-40 buffer (40 mM HEPES, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 8.0; 1% NP-40 (CA-630, Sigma); 5% glycerol; 10 mM pyrophosphate; 10 mM β-glycerophosphate; 50 mM NaF; 0.5 mM orthovanadate) containing Protease Inhibitor Cocktail (Sigma) and 1 mM DTT. Nuclear isolation was performed with a Nuclear Extract Kit (40010, Active Motif, Carlsbad, CA, USA), with 10 μg/ml ALLN (208719, Millipore, Bedford, MA, USA) treatment 20 min prior to isolation, and ALLN added to the hypotonic and lysis buffers. The nuclear fraction was washed with hypotonic buffer prior to lysis.
Antibodies used for immunoblots recognized SREBP1 (sc-8984, Santa Cruz, Santa Cruz, CA, USA), SREBP2 precursor and processed C-terminus (557037, BD, Franklin Lakes, NJ, USA), SREBP2 mature N-terminus (30682, Abcam, Cambridge, MA, USA), Actin (A5316, Sigma), and from Cell Signaling Technologies (Danvers, MA, USA): ACC1 (3676), FASN (3180), SCD (2438), HA (2367), P-Akt-T308 (9275), P-Akt-S473 (4051), Total-Akt (4691), P-S6K1-T389 (9234), Total-S6K1 (2708), P-S6-S240/S244 (2215), Total-S6 (2217), 4E-BP1 (9644), Ras (3965), P-Erk1/2-T202/Y204 (9106), Total-Erk1/2 (9102), Lamin A/C (2032), Histone H3 (4499).

Ricoult S.J., Yecies J.L., Ben-Sahra I, & Manning B.D. (2015). Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP. Oncogene, 35(10), 1250-1260.

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Antibody Panel for Cell Signaling

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eIF2α, P-eIF2α (Ser51), Total S6K1, P-S6K1 (T389), Cleaved PARP, total and cleaved caspase-3 antibodies were purchased from Cell Signaling Technologies, ATF4 (SC-200) from Santa Cruz and MAP1LC3B antibody from Novus. Antibody Puromycin (clone 12D10) was kindly given by Phillipe Pierre (Centre d'Immunologie de Marseille-Luminy, France).

Mesclon F., Lambert-Langlais S., Carraro V., Parry L., Hainault I., Jousse C., Maurin A.C., Bruhat A., Fafournoux P, & Averous J. (2017). Decreased ATF4 expression as a mechanism of acquired resistance to long-term amino acid limitation in cancer cells. Oncotarget, 8(16), 27440-27453.

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Protein Extraction and Immunoblotting Protocol

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Cells were lysed in ice-cold NP-40 lysis buffer (40 nM HEPES [pH 7.4], 400 nM NaCl, 1 mM EDTA [pH 8.0], 1% NP-40 [CA-630, Sigma-Aldrich], 5% glycerol, 10 mM pyrophosphate, 10 mM β-glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate) containing protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1 µM microcystin-LR (ALX-350–012-C500, Enzo Life Sciences, Farmingdale, NY, USA). Lysates were clarified by centrifugation (20,000 × g for 15 min at 4 °C) and protein concentrations were determined with Bradford assay (Bio-Rad) prior to normalization. The following antibodies were used for detection of proteins transferred to immobilon-P PVDF membranes after SDS-PAGE: β-actin (A5316, Sigma-Aldrich), tubulin (T5168, Sigma-Aldrich), TFEB (A303-673A, Bethyl, Montgomery, TX, USA), HIF1-α (10006421, Cayman Chemical, Ann Arbor, MI, USA), SREBP1 (for human samples; sc-8984, Santa Cruz), SREBP1 (for mouse samples; 557036, BD Biosciences), PIM1 (sc-13513, Santa Cruz). All other antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA): PIM3 (4165), SCD (2438), P-S6K1-T389 (9234), Total-S6K1 (2708), ATF4 (11815), and c-Myc (13987).

Kelsey I., Zbinden M., Byles V., Torrence M, & Manning B.D. (2017). mTORC1 suppresses PIM3 expression via miR-33 encoded by the SREBP loci. Scientific Reports, 7, 16112.

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Investigating Skeletal Muscle Protein Signaling

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Both primary and secondary antibodies were diluted with TBS containing 2.5% milk. The primary antibodies used were Mouse Anti-Slow Skeletal Myosin Heavy Chain (Abcam #ab11083, diluted 1:10,000), Rabbit Anti-Fast Myosin Skeletal Heavy Chain (Abcam #ab91506, diluted 1:10,000), total mTOR (Cell Signaling #2983S, diluted 1:1,000), phosphorylated mTOR Ser²⁴⁴⁸ (Cell Signaling #5536S, diluted 1:1,000), total S6K1 (Cell Signaling #2708S, diluted 1:1,000), phosphorylated S6K1 Thr³⁸⁹ (Cell Signaling #9234S, diluted 1:1,000), total eEF2 (Cell Signaling #2332S, diluted 1:1,000), and phosphorylated eEF2 Thr⁵⁶ (Cell Signaling #2331S, diluted 1:1,000). The secondary antibodies, anti-rabbit HRP-linked (#7074S) and anti-mouse HRP-linked (#7076S), were both purchased from Cell Signaling and diluted 1:10,000.

Edman S., Söderlund K., Moberg M., Apró W, & Blomstrand E. (2019). mTORC1 Signaling in Individual Human Muscle Fibers Following Resistance Exercise in Combination With Intake of Essential Amino Acids. Frontiers in Nutrition, 6, 96.

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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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GSK621 Autophagy Regulation Assay

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GSK621 was purchased from Gao-Chem (Shanghai, China). H₂O₂, puromycin, 3-methyaldenine (3-MA) and chloroquine (Cq) were purchased from Sigma Chemicals (St. Louis, MO). Anti-AMPKα1, acetyl-CoA carboxylase (ACC) and Beclin-1, autophagy-related homologue 5 (ATG-5), light chain 3B-II (LC3B-II), p62 and Erk1/2 antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). Antibodies against phospho(p)-AMPKα1 (Thr 172), p-ACC (Ser 79), p-S6K1 (Thr-389), p-S6 (Ser-235/236) and total S6K1 were obtained from Cell Signaling Tech (Denver MA).

Liu W., Mao L., Ji F., Chen F., Hao Y, & Liu G. (2017). Targeted activation of AMPK by GSK621 ameliorates H2O2-induced damages in osteoblasts. Oncotarget, 8(6), 10543-10552.

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Western Blot Antibody Validation Protocol

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The following antibodies were used for western blots: from Cell Signaling, Danvers, MA: total mTOR (1:1000; catalog number 2983S), total S6K1 (1:1000; catalog number 9202S), pS6K1 Thr389 (1:1000; catalog number 9205S), β-actin (1:10000; catalog number 3700S), total IRS-1 (1:1000; catalog number 3407S), IRS Ser318 (1:1000; catalog number 5610S), IRS Ser636/639 (1:1000; catalog number 2388S), totalPI3K p85 (1:1000; catalog number 4292S), PI3K Tyr458 (1:1000; catalog number 4228S), total AKT (1:1000 ; catalog number 9272), AKT Thr308 (1:1000; catalog number 2965S), AKT Ser473 (1:1000; catalog number3787S) total GSK3β (1:1000; catalog number 9315S), GSK3β Ser9 (1:1000; catalog number 9336S), CP13 (1:1000; catalog number 11834S). From Millipore, Billerica, MA: APP C-terminal (1:2000; catalog number 171610), Tau5 (1:5000; catalog number 577801). From BioLegend, San Diego, CA: APP (1:3000; catalog number MAB348). PHF-1 (1:3000) was a gift from Dr. Peter. Davies.

Caccamo A., Belfiore R, & Oddo S. (2018). Genetically reducing mTOR signaling rescues central insulin dysregulation in a mouse model of Alzheimer’s disease. Neurobiology of aging, 68, 59-67.

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Immunoblot Analysis of Cellular Signaling

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Whole cell extracts were prepared in 1% SDS solution containing protease inhibitors. Equal amounts of proteins from the cell lysates were separated by SDS-PAGE, followed by immunoblot analyses as previously described (28 (link)). Proteins bound to membrane filters were probed with the specified antibodies and immunoreactive proteins were visualized using chemiluminiscence using either X-ray film or the Bio-Rad Chemidoc MP imaging system. Antibodies using in the immunoblot analyses include the following: P-GCN2 (Abcam, #Ab75836), total GCN2 (Abcam, #ab137543), P-eIF2α (Abcam, #ab32157), total eIF2α (Cell Signaling Technology, #5324S), ATF4 antibody was prepared against the corresponding recombinant human proteins, which were affinity purified (30 (link)), P-mTOR (Cell Signaling Technology, #2971S), total mTOR (Cell Signaling Technology, #2983S), P-S6K1 (Cell Signaling Technology, #9205S), total S6K1 (Cell Signaling Technology, #9202S), P-4EBP1 (Cell Signaling Technology, #2855S), total 4EBP1 (Cell Signaling Technology, #9644S), PRODH (Proteintech, # 22980-1-AP) and β-actin (Sigma, #A5441). Quantification of immunoblots was carried out by measuring relative band intensities measured using ImageJ software, which was normalized as indicated.

Misra J., Holmes M.J., T. Mirek E., Langevin M., Kim H.G., Carlson K.R., Watford M., Dong X.C., Anthony T.G, & Wek R.C. (2021). Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress. Nucleic Acids Research, 49(10), 5726-5742.

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Western Blot Analysis of Metabolic Proteins

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Cells were harvested and lysed in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 3% sodium dodecyl sulfate (SDS), 10% glycerol, 50 mM DL-dithiothreitol (DTT), and 0.1% bromophenol blue) with protease inhibitors (Roche, Indianapolis, IN, USA). The protein concentrations were determined by the BCA method (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA). The proteins (30 μg) were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Bovine serum albumin (5%) in TBS-T (1 mol/L Tris-HCl (pH 7.5), 0.8% NaCl and 0.1%Tween 20) was used to block the membrane. Then, the membrane was incubated with anti-Glut1 (Santa-Cruz, sc-7903, CA, USA), anti-LDHA (Santa-Cruz, sc-137243, CA, USA), anti-phosphor-S6K1 (Cell Signaling, #9209) and total S6K1 (Cell Signaling, #9202), anti-phosphor-AKT (Cell Signaling, #13038) and total Akt (Cell Signaling, #4691) and anti-β-actin (Sigma-Aldrich, A5441, St.Louis.USA) antibodies at 4°C overnight. The blots were then treated with an HRP-conjugated secondary antibody (Pierce)

Zou Z.W., Ma C., Medoro L., Chen L., Wang B., Gupta R., Liu T., Yang X.Z., Chen T.T., Wang R.Z., Zhang W.J, & Li P.D. (2016). LncRNA ANRIL is up-regulated in nasopharyngeal carcinoma and promotes the cancer progression via increasing proliferation, reprograming cell glucose metabolism and inducing side-population stem-like cancer cells. Oncotarget, 7(38), 61741-61754.

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Western Blot Analysis of mTOR Signaling

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Cells were lysed in lysis buffer (20 nM Tris, pH 7.5, 135 mM NaCl, 5% [v/v] glycerol, 50 nM NaF, 0.1% Triton X‐100), as described previously (Dunlop et al., 2014 (link)), with added protease inhibitors. Following sonication, centrifugation and protein quantification, samples were prepared in 4X LDS sample buffer (Invitrogen) with 25 mM dithothreitol (DTT). Lysates were separated by a 4%–12% gradient gel (Invitrogen), transferred onto PVDF membranes, blocked, then incubated with primary antibodies against TSC2 (catalogue #3990), total S6K1 (catalogue #9202), phospho‐S6K1 (T389) (catalogue #9205), pan‐Akt (catalogue #4691), phospho‐Akt (S473) (catalogue #4060) (all Cell Signaling Technology), ALIX (catalogue #sc‐166952), TSG101 (catalogue #sc‐7964), GRP94 (catalogue #sc‐393402) (all Santa Cruz), GAPDH (Novus Biologicals catalogue #1A10), overnight at 4°C. Following washing, secondary antibody incubation and further washes, proteins were visualised using LI‐COR ECL Reagent and a C‐DiGit® Blot Scanner (both LI‐COR Biotechnology) or Amersham ECL reagent and an ImageQuant 800 imaging system (both Cytiva).

Bhaoighill M.N., Falcón‐Pérez J.M., Royo F., Tee A.R., Webber J.P, & Dunlop E.A. (2023). Tuberous Sclerosis Complex cell‐derived EVs have an altered protein cargo capable of regulating their microenvironment and have potential as disease biomarkers. Journal of Extracellular Vesicles, 12(6), 12336.

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