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Total s6k

Manufactured by Cell Signaling Technology
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Total S6K is a lab equipment product that measures the total level of S6 Kinase (S6K), an important signaling protein involved in cell growth and metabolism. It provides a quantitative assessment of the total S6K content in a sample, without differentiating between the active and inactive forms of the protein.

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Lab products found in correlation

Total akt, by Cell Signaling Technology (12 mentions) P s6kt389, by Cell Signaling Technology (4 mentions) Pakt s473, by Cell Signaling Technology (4 mentions) P s6k, by Cell Signaling Technology (4 mentions) β actin, by Merck Group (3 mentions) Phospho akt ser473, by Cell Signaling Technology (3 mentions) P akt, by Cell Signaling Technology (3 mentions) P akt ser473, by Cell Signaling Technology (2 mentions) P mtor ser2448, by Cell Signaling Technology (2 mentions) Raptor, by Cell Signaling Technology (2 mentions) Cd41 fitc, by BioLegend (2 mentions) Annexin 5 fitc, by BioLegend (2 mentions) Human insulin elisa kit, by ALPCO (2 mentions) Rat insulin elisa kit, by ALPCO (2 mentions) Pakt t308, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Mouse ultrasensitive insulin elisa, by ALPCO (2 mentions) Human rcela2a, by MyBioSource (2 mentions) Cela2a elisa kit, by MyBioSource (2 mentions) Cela2a antibody, by Merck Group (2 mentions)

Close spelling variants of this product

Anti-total S6K (4 citations) PathScan® Total S6K Sandwich ELISA Kits (2 citations) Rb anti-total p70 S6K (2 citations)

16 protocols using total s6k

1

Immunoblotting and Immunohistochemistry Protocols

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The following primary antibodies were used in this study for immunoblotting: pRBser780 (CST-3590), pRBser807 (CST-8516), total-RB (CST-9309), cyclin D1 (CST-2922), cyclin D3 (CST-2936), pAKTser473 (CST-9271), pAKTThr308 (CST-9275), total-AKT (CST-9272), pEGFRTyr1068 (CST-3777), total-EGFR (CST-2232), pERBB2Tyr1248 (CST-2243), total-ERBB2 (CST-4290), pERBB3Tyr1222 (CST-4784), pIGF1RTyr1135 (CST-3918), pS6KSer235/236 (CST-2211), total-S6K (CST-2217), Raptor (CST-2280), RheB (CST-13879), p4EBP1Thr37/46 (CST-2855), 4EBP1 (CST-9452), pSIN1Thr86 (CST-14716), SIN1 (CST-12860), pERser167 (CST-5587), Rictor (CST-2114) and Deptor (SCT-11816) were purchased from Cell Signaling Technology. p107 (sc-318), p130 (sc-317), total-ER (sc-8002, F-10), ERBB3 (sc-415) and IGF1R (sc-713) were purchased from Santa Cruz Biotechnology. β-tubulin (T-9026) were from Sigma-Aldrich and Ki67 from Clinisciences. The following antibodies were used for immunohistochemistry: pERK1/2Thr202/4 (CST-4370), pAKTser473 (CST-4060), pS6KSer235/6 (CST-4858), pmTORSer2448 (CST-2976) and p4EBP1Thr37/46 (CST-2855) were purchased from Cell Signaling Technology. Ki67 was purchased from Clinisciences. Reagents were obtained from the following sources: 17-β-oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) from Sigma-Aldrich, fulvestrant from Tocris, and neratinib and vistusertib from SelleckChem.
Pancholi S., Leal M.F., Ribas R., Simigdala N., Schuster E., Chateau-Joubert S., Zabaglo L., Hills M., Dodson A., Gao Q., Johnston S.R., Dowsett M., Cosulich S.C., Maragoni E, & Martin L.A. (2019). Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer. Breast Cancer Research : BCR, 21, 135.
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2

Quantitative Platelet and Pancreatic Assays

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Purified GP2B3A complex (MBS135714), mouse rCela2a protein (MBS1246487), human rCELA2A (MBS1090462) and the CELA2A ELISA kit (MBS932150) were purchased from MyBiosource. CELA2A antibody (SAB1104798) and the elastase substrate N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide substrate (S8511) were purchased from Sigma-Aldrich. The FITC-conjugated PAC1 antibody (340507, BD Biosciences) and tetramethylrhodamine ethyl ester (TMRE) reagent (T669, Thermo Fisher Scientific), Annexin V-FITC (640905, Biolegend), CD41-FITC (303703, Biolegend) were used for platelet aggregation analysis. Total AKT (CST, 9272), pAKT (S473) (CST, 9271s), pAKT (T308) (CST, 9275s), total S6k (CST, 9202), pS6k (T389) (CST, 9206), total IRS (CST, 2382), pIRS (Y608), (CST, 2385S), pIRS (S636/S639) (CST, 2388s), Gapdh (CST, 3683s), and Actin (CST, 4970) antibodies were purchased from Cell Signaling Technologies. Recombinant A1AT (alpha1 antitrypsin, ab91136), A1AT antibody (ab9400) and ITGA2B antibodies (ab63983) were purchased from Abcam. Human c-peptide ELISA kit (80-CPTHU-E01.1, ALPCO), Human Glucagon ELISA kit (10-1271-01, Mercodia), Rat c-peptide ELISA (80-CPTRT-E01, ALPCO), Mouse Ultrasensitive Insulin ELISA (80-INSMSU-E01, ALPCO), Human Insulin ELISA kit (21-IAAHU-E01, ALPCO), Rat Insulin ELISA kit (80-INSRT-E01, ALPCO) was applied for C-peptide, glucagon, and insulin measurements.
Esteghamat F., Broughton J.S., Smith E., Cardone R., Tyagi T., Guerra M., Szabó A., Ugwu N., Mani M.V., Azari B., Kayingo G., Chung S., Fathzadeh M., Weiss E., Bender J., Mane S., Lifton R.P., Adeniran A., Nathanson M.H., Gorelick F.S., Hwa J., Sahin-Tóth M., Belfort-DeAguiar R., Kibbey R.G, & Mani A. (2019). CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nature genetics, 51(8), 1233-1243.
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3

Immunoblotting of Cellular Signaling Proteins

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Rabbit anti-human primary antibodies: Total S6K (1:1000; Cell Signaling, #2708), p-Thr389 S6K pAb (1:1000; EMD Millipore, #07-018-I), Total Akt (1:1000; Cell Signaling, #4691), p-Ser473 Akt (1:2000; Cell Signaling, #4060), total mTOR (1:1000; Cell Signaling, #2983), p-Ser2448 mTOR (1:1000; Cell Signaling, #5536), Alexa Fluor 790 to GAPDH (1:1000; Abcam loading control, #ab184578). Secondary antibody: HRP-linked anti-rabbit IgG (1:3000; Cell Signaling, #7074 S).
Belliveau N.M., Footer M.J., Akdoǧan E., van Loon A.P., Collins S.R, & Theriot J.A. (2023). Whole-genome screens reveal regulators of differentiation state and context-dependent migration in human neutrophils. Nature Communications, 14, 5770.
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4

Quantitative Platelet and Pancreatic Assays

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Purified GP2B3A complex (MBS135714), mouse rCela2a protein (MBS1246487), human rCELA2A (MBS1090462) and the CELA2A ELISA kit (MBS932150) were purchased from MyBiosource. CELA2A antibody (SAB1104798) and the elastase substrate N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide substrate (S8511) were purchased from Sigma-Aldrich. The FITC-conjugated PAC1 antibody (340507, BD Biosciences) and tetramethylrhodamine ethyl ester (TMRE) reagent (T669, Thermo Fisher Scientific), Annexin V-FITC (640905, Biolegend), CD41-FITC (303703, Biolegend) were used for platelet aggregation analysis. Total AKT (CST, 9272), pAKT (S473) (CST, 9271s), pAKT (T308) (CST, 9275s), total S6k (CST, 9202), pS6k (T389) (CST, 9206), total IRS (CST, 2382), pIRS (Y608), (CST, 2385S), pIRS (S636/S639) (CST, 2388s), Gapdh (CST, 3683s), and Actin (CST, 4970) antibodies were purchased from Cell Signaling Technologies. Recombinant A1AT (alpha1 antitrypsin, ab91136), A1AT antibody (ab9400) and ITGA2B antibodies (ab63983) were purchased from Abcam. Human c-peptide ELISA kit (80-CPTHU-E01.1, ALPCO), Human Glucagon ELISA kit (10-1271-01, Mercodia), Rat c-peptide ELISA (80-CPTRT-E01, ALPCO), Mouse Ultrasensitive Insulin ELISA (80-INSMSU-E01, ALPCO), Human Insulin ELISA kit (21-IAAHU-E01, ALPCO), Rat Insulin ELISA kit (80-INSRT-E01, ALPCO) was applied for C-peptide, glucagon, and insulin measurements.
Esteghamat F., Broughton J.S., Smith E., Cardone R., Tyagi T., Guerra M., Szabó A., Ugwu N., Mani M.V., Azari B., Kayingo G., Chung S., Fathzadeh M., Weiss E., Bender J., Mane S., Lifton R.P., Adeniran A., Nathanson M.H., Gorelick F.S., Hwa J., Sahin-Tóth M., Belfort-DeAguiar R., Kibbey R.G, & Mani A. (2019). CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nature genetics, 51(8), 1233-1243.
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5

Quantitative Western Blot Analysis

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Western blot experiments were conducted with antibodies raised against LC3 (1:4000; Sigma-Aldrich, #L7543), p62 [1:1000; Enzo Life Sciences, #BML-PW9860-0025; similar data were obtained in pilot experiments when a different anti-p62 antibody was used (1:1000; Progen, Heidelberg, Germany, #GP62-C)], phosphorylated S6K-T389 (1:1000; Cell Signaling, #9206), total S6K (1:1000; Cell Signaling, #9202), phosphorylated ULK1-S757 (1:1000; Cell Signaling, #6888), phosphorylated ULK1-S555 (1:1000; Cell Signaling, #5869), total ULK1 (1:1000; Cell Signaling, #8054) or β-actin (1:4000, Sigma-Aldrich, #A5441), followed by the corresponding HRP-linked secondary antibodies (1:5000; GE-Healthcare, Madrid, Spain, #NA931; GE-Healthcare, #NA934; Invitrogen, #A18769), as appropriate. Densitometric analysis was performed with Image J software (NIH).
Blázquez C., Ruiz-Calvo A., Bajo-Grañeras R., Baufreton J.M., Resel E., Varilh M., Pagano Zottola A.C., Mariani Y., Cannich A., Rodríguez-Navarro J.A., Marsicano G., Galve-Roperh I., Bellocchio L, & Guzmán M. (2020). Inhibition of striatonigral autophagy as a link between cannabinoid intoxication and impairment of motor coordination. eLife, 9, e56811.
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6

Investigating PI3K/mTOR Inhibitor Effects

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SKLMS1 and STS39 cells were seeded in six well plates at a density of 250,000 c/well and 500,000 c/well respectively. Cells were treated with BKM120 or BEZ235 (5–1000 nM) for 72 h prior to harvest. Following a cold PBS (Sigma) rinse, cells were lysed for 20 min on ice with RIPA buffer (50 mM pH 7.4 Tris–HCl, 150 mM NaCl, 1 % NP-40, 1 mM EDTA) supplemented with phosphatase and protease inhibitors (Sigma). Protein concentration was measured with DC Protein concentration assay (BioRad). Electrophoresis was performed using MiniProtean TGX gels (Bio-Rad) and transferred to PVDF by wet transfer. Immunoblots were performed with the following antibodies: p-AKTS473, total AKT, p-S6KT389, total S6K, p-4EBP1T37/46, total 4EBP1, PARP-1, all from Cell Signaling Technology (Denver, US), and tubulin [clone DM1A] (Sigma, St. Louis, USA). All immunoblots shown are representative of at least three independent experiments.
Babichev Y., Kabaroff L., Datti A., Uehling D., Isaac M., Al-awar R., Prakesch M., Sun R.X., Boutros P.C., Venier R., Dickson B.C, & Gladdy R.A. (2016). PI3K/AKT/mTOR inhibition in combination with doxorubicin is an effective therapy for leiomyosarcoma. Journal of Translational Medicine, 14, 67.
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7

Western Blot Analysis of Vascular Signaling

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Aortic rings and endothelial positive and negative cell separations were homogenized in RIPA buffer (#R0278; Sigma) and a protease inhibitor cocktail (#11836170001; Roche). Lysates were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes and probed for Raptor (1:1000; Cell Signaling #2280), eNOS (endothelial nitric oxide; 1:1000; BD Biosciences #610297), phospho-eNOS (Ser1177; 1:1000; Cell Signaling #9571), phospho-S6 (1:5000; Cell Signaling #5364), total S6 (1:1000; Cell Signaling #2217), total S6K (1:1000; Cell Signaling #2708), NRF2 (nuclear factor erythroid 2–related factor 2; 1:1000; Abcam #ab62352), p62 (1:1000; Cell Signaling #5114), LC3A (1:1000; Cell Signaling #4599), and β-actin (1:50000; Proteintech #60008). Protein detection was performed using HRP-conjugated anti-rabbit (1:10000; Cell Signaling #7074) or anti-mouse (1:10000; Cell Signaling #7076) secondary antibodies and visualized either on film with an ECL Prime chemiluminescent kit (GE Healthcare) or imaged on a Sapphire Biomolecular Imager (Azure Biosystems). Densitometry was calculated with ImageJ software.
Reho J.J., Guo D.F., Morgan D.A, & Rahmouni K. (2020). Mechanistic Target of Rapamycin Complex 1 (mTORC1) Signaling in Endothelial and Smooth Muscle Cells is Required for Vascular Function. Hypertension (Dallas, Tex. : 1979), 77(2), 594-604.
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8

Insulin Signaling Pathway Analysis

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S2 cells were serum starved for 2 hr, treated with trametinib dissolved in DMSO or DMSO alone for 30 min, and then stimulated with 1 μM human insulin (Sigma) for 15 min. Western blots were probed for phospho-Erk(Thr202/Tyr204) (#4370), phospho-Akt(Ser473) (#4060), phospho-S6K(Thr398) (#9209), total-Erk (#4695), total-Akt (#9272) (Cell Signaling Technologies), total-S6K, and tubulin.
Slack C., Alic N., Foley A., Cabecinha M., Hoddinott M.P, & Partridge L. (2015). The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for Longevity. Cell, 162(1), 72-83.
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9

Cardiac Protein Analysis via Western Blot

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Total protein lysates were extracted from frozen hearts as described previously [21 (link)]. Isolated cardiomyocytes were isolated and lysates prepared for western blot as previously described by us [22 (link)]. Protein extracts were resolved by SDS-PAGE and electro-transferred onto PVDF membranes (Millipore Corp., Billerica, MA). The following antibodies were used: phospho-Akt-Ser 473, phospho-Akt-Thr 308, total Akt, phospho-p70 S6K and total S6K (Cell Signaling, Danvers, MA). To control for loading, mouse anti-GAPDH and anti-actin antibodies (Sigma, Saint Louis, MO) were used. Alexa fluor anti-Rabbit 680 (Invitrogen, Carlsbad, CA) and Mouse 800 (VWR International, West Chester, PA) were used as secondary antibodies and fluorescence was quantified using the LI-COR Odyssey imager (Lincoln, NE).
Noh J., Wende A.R., Olsen C.D., Kim B., Bevins J., Zhu Y., Zhang Q.J., Riehle C, & Abel E.D. (2015). Phosphoinositide Dependent Protein Kinase 1 is Required for Exercise-induced Cardiac Hypertrophy but not the Associated Mitochondrial Adaptations. Journal of molecular and cellular cardiology, 89(0 0), 297-305.
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10

Western Blot Analysis of Cellular Signaling

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Cells were lysed in modified RIPA buffer containing 50 mmol/L Tris-HCl (pH, 8), 300 mmol/L NaCl, 10% NP-40, 1% sodium deoxycholate, and 0.1% SDS and a protease inhibitor cocktail tablet (Roche Applied Science). Protein extracts, approximately 30 μg of each sample, were resolved by SDS-PAGE followed by immunoblotting with ARK5 antibody (Cell Signaling Technology #4458), total MYC (Cell Signaling Technology #9402), acetyl MYC lysine 323 (Millipore #ABE26), SIRT1 (Cell Signaling Technology # 2310), AMPK (Cell Signaling Technology #2532), total Rb (Santa Cruz Biotechnology #sc-74562), phosphoRb (Cell Signaling Technology #9308), total S6K (Cell Signaling Technology #9202), phosphoS6K (Cell Signaling Technology #9205), and actin antibody (C-11, horseradish peroxidase, goat polyclonal antibody; Santa Cruz Biotechnology) and detected by enhanced chemiluminescence (Santa Cruz Biotechnology). After treatment, cells were harvested and washed with ice-cold PBS and subsequently lysed with RIPA buffer with fresh protease and phosphatase inhibitors. Blot patterns were analyzed using ImageJ software (http://rsbweb.nih.gov/ij/), providing a quantitative measure of protein expression.
Perumal D., Kuo P.Y., Leshchenko V.V., Jiang Z., Divakar S.K., Cho H.J., Chari A., Brody J., Reddy M.V., Zhang W., Reddy E.P., Jagannath S, & Parekh S. (2016). Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma. Cancer research, 76(5), 1225-1236.
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