Reagents were obtained from the following sources. Antibodies to TSC2 (#4308), phospho-T389 S6K1 (#9234), phospho-S240/S244 S6 (#5364), phospho-T980 PERK (#3179), PERK (#3192), Psma5 (#2457), Psmb5 (#11903), Psmd14 (#4197), FLAG (#2368), normal rabbit IgG (#2729), and horseradish-peroxidase-labelled anti-rabbit (#7074S) and anti-mouse (#7076S) secondary antibodies from Cell Signaling Technology; antibodies to NRF1 (sc-13031) and SREBP1 (sc-8984) from Santa Cruz; antibody to NRF2 (#2178-1) and Psma1 (3759-1) from Epitomics; antibodies to β-actin (A-5316) and α-tubulin (T-5168) from Sigma. 35S-methionine, cell labeling grade, from PerkinElmer Life Sciences; L-methionine, chloroquine, MG132, tunicamycin, thapsigargin, cycloheximide, trichloroacetic acid, sodium salicylate, protease inhibitor cocktail, Dulbecco’s modified Eagle’s medium (DMEM)/F12, insulin, hydrocortisone, cholera toxin, and fetal bovine serum from Sigma; methionine-free DMEM, Basal Medium Eagle (BME), 50X amino acids solution, horse serum, transfection reagents Lipofectamine® 2000 and Lipofectamine® RNAiMAX, and NuPAGE® Novex® 4-12% Bis-Tris protein gel from Life Technologies Corporation; rapamycin and bortezomib from LC Laboratories; Torin1 and PP242 from Tocris; EGF from Peprotech.
Phospho s6k1 t389
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-S6K1 T389 is a lab equipment product that detects the phosphorylation of S6 kinase 1 (S6K1) at threonine 389. It is used to measure the activation status of the mTOR signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Total s6k1, by Cell Signaling Technology (2 mentions)
L methionine, by PerkinElmer (1 mentions)
Psmd14, by Cell Signaling Technology (1 mentions)
Nupage novex 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem f12, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Cholera toxin, by Merck Group (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Bortezomib, by LC Laboratories (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Evagreen, by Biotium (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
5 protocols using phospho s6k1 t389
1
Quantitative Proteomic Analysis of mTOR Signaling
2
Antibody Sourcing and Western Blot Protocol
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Reagents were obtained from the following sources. Monoclonal antibody phospho-T389 S6K1, rabbit antibody S6K, and rabbit antibody ATF4, which could react with antigens from rat, were obtained from Cell Signaling Technology. Rabbit antibody LC3 was obtained from MBL, rabbit antibody β-actin was obtained from Sigma-Aldrich, and rabbit antibody SLC7A5 was obtained from Cosmo Bio Company and could react with antigens from rat. 2-Aminobicyclo-(2, 2, 1)-heptane-2-carboxyl acid was obtained from Sigma-Aldrich. EvaGreen was obtained from Biotium and Trizol from Invitrogen. RNeasy Mini kit was purchased from QIAGEN, RevertAid First Strand cDNA Synthesis kit from Fermentas, BCA Protein Assay kit from Thermo Fisher Scientific, and Fluoromount-G from SouthernBiotech. Western blots were performed as described previously (Yu et al., 2010 (link)).
3
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).
4
Immunoblotting of Dystrophin and S6K1
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Mouse anti-dystrophin, developed by Morris, G.E. (DSHB Hybridoma Product MANDRA1(7A10)), was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA 52242. Phospho-S6K1 (T389, Cat#: 9205) and phospho-S6K1 (T421/S424, Cat#: 9204) were obtained from Cell Signaling Technology (Danvers, MA, USA). S6K1 (Cat#: sc-230) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IRDye 680LT Goat anti-Rabbit IgG (Cat#: 926–68021) was from LI-COR Biosciences (Lincoln, NE, USA). Alexa Fluor 568-conjugated Goat anti-Mouse IgG (Cat#: A11031) was from Thermo Fisher Scientific (Rockford, IL, USA).
5
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).
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