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Total p70s6k

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Total p70S6K is an ELISA kit designed to quantitatively measure the total amount of p70S6K protein in cell or tissue lysates. p70S6K is a serine/threonine protein kinase that plays a key role in the regulation of cell growth and proliferation.

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Lab products found in correlation

Total akt, by Cell Signaling Technology (8 mentions) Total erk1 2, by Cell Signaling Technology (5 mentions) Phospho p70s6k, by Cell Signaling Technology (5 mentions) Total 4e bp1, by Cell Signaling Technology (5 mentions) P p70s6k, by Cell Signaling Technology (4 mentions) β actin, by Merck Group (4 mentions) Phospho p70s6k thr389, by Cell Signaling Technology (4 mentions) Phosphorylated p70s6k, by Cell Signaling Technology (4 mentions) β actin, by Cell Signaling Technology (4 mentions) Total s6, by Cell Signaling Technology (3 mentions) P akt, by Cell Signaling Technology (3 mentions) Phospho 4ebp1, by Cell Signaling Technology (3 mentions) Phospho erk1 2, by Cell Signaling Technology (3 mentions) P erk1 2, by Cell Signaling Technology (2 mentions) Phospho akt, by Cell Signaling Technology (2 mentions) Phospho s6, by Cell Signaling Technology (2 mentions) Cyclin d1, by Cell Signaling Technology (2 mentions) Total acc, by Cell Signaling Technology (2 mentions) Phospho p70s6k t389, by Cell Signaling Technology (2 mentions) Total erk, by Cell Signaling Technology (2 mentions)

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P70S6K (353 citations) Anti-total p70S6K (5 citations) PathScan total P70S6K plates (2 citations)

25 protocols using total p70s6k

1

Western Blot Analysis of Cell Signaling Proteins

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Western blot analysis was performed essentially as described [19 (link)]. The method used to normalize the protein levels was “Pierce BCA Protein Assay Kit” (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (15 μg total protein per lane) were prepared in sodium dodecyl sulfate (SDS) sample buffer, separated by SDS-PAGE, and blotted on polyvinylidene difluoride membranes. The following antibodies were used: mouse anti-human β-actin (Proteintech, IL, USA), perilipin (Cell Signaling, MA, USA), phosphor-P70S6K (Cell Signaling, MA, USA), total P70S6K (Cell Signaling, MA, USA), pERK1/2 (Cell Signaling, MA, USA), total ERK1/2 (Cell Signaling, MA, USA), anti-mouse IgG HRP conjugate (Proteintech, IL, USA), and rabbit anti-rat IgG HRP (Proteintech, IL, USA). Image J software was used for densitometric analyses.
Chinnapaka S., Yang K.S., Flowers Q., Faisal M., Nerone W.V., Rubin J.P, & Ejaz A. (2021). Metformin Improves Stemness of Human Adipose-Derived Stem Cells by Downmodulation of Mechanistic Target of Rapamycin (mTOR) and Extracellular Signal-Regulated Kinase (ERK) Signaling. Biomedicines, 9(12), 1782.
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2

Western Blot Protein Expression Analysis

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Cells were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% NP-40; 50 mM NaF with protease inhibitors) and incubated on ice for 30 min. Lysates were centrifuged at 20,817g at 4 °C for 10 min and supernatant was collected. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein samples were mixed with SDS Laemmli loading buffer, boiled and electrophoresed using NuPAGE Bis-Tris Gels (Life Technologies), then transferred onto PVDF membranes (Millipore). Blocking was performed for 45 min using TBST supplemented with 5% non-fat dry milk and blotting performed with primary antibodies at 4 °C for 16 h. The following antibodies were used: ADSL (Abcam #ab154182), GMPS (Cell Signaling #14602), PRPS1 (Abcam #ab154721), MYC (N-262, Santa Cruz #sc-764), total AKT (Cell Signaling #4691), phospho-AKT (S473, Cell Signaling #9271), total S6 (Cell Signaling #2317), phospho-S6 (235/236, Cell Signaling #4858), phospho-S6 (240/242, Cell Signaling #5364), total p70 S6K (Cell Signaling #2708), phospho-p70 S6K (Cell Signaling #9234) and α-tubulin (Sigma #T6074). All antibody validation information is available in the product’s manual. For western blotting, the dilution was 1:500 for MYC antibody and 1:1,000 for all other antibodies.
Wang X., Yang K., Xie Q., Wu Q., Mack S.C., Shi Y., Kim L.J., Prager B.C., Flavahan W.A., Liu X., Singer M., Hubert C.G., Miller T.E., Zhou W., Huang Z., Fang X., Regev A., Suvà M.L., Hwang T.H., Locasale J.W., Bao S, & Rich J.N. (2017). Purine synthesis promotes maintenance of brain tumor initiating cells in glioma. Nature neuroscience, 20(5), 661-673.
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3

Analyzing Cell Signaling Pathways

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Axin2 (#2151, Cell signaling technology), c-Myc (#1472–1, Epitomics), β-actin (#A1978, Sigma), pACC (#11818, Cell signaling technology), Total ACC (#3676, Cell signaling technology), pAMPK (#2535, Cell signaling technology), Total AMPK (#2532, Cell signaling technology), GAPDH (#GTX627408, GeneTex), Plk1 (#sc-17783, Santa Cruz), cyclinD1 (#2978, Cell signaling technology), cyclinB1 (#4135, Cell signaling technology), p-Akt (#9272, Cell signaling technology), Total-Akt (#1081–1, Epitomics), p-P70S6K (#9234, Cell signaling technology), Total-P70S6K (#2708, Cell signaling technology), p-4E-BP1 (#2855, Cell signaling technology), Total-4E-BP1 (#GTX109162, GeneTex), p-CDC2 (#9111, Cell signaling technology), Total-CDC2 (#GTX108120, GeneTex). Compounds: GSK461364 was purchased from Cayman Chemical (Ann Arbor, MI, USA). BI2536 was from Achemblock (Burlingame, CA). DAPI and PI were from ThermoFisher (Waltham, MA).
Xie Y., Zhang W., Guo L., Kril L.M., Begley K.L., Sviripa V.M., Chen X., Liu X., Lee E.Y., He D., Wang C., Gao T., Liu X., Evers B.M., Watt D.S, & Liu C. (2021). Potent Synergistic Effect on c-Myc Driven Colorectal Cancers Using a Novel Indole-Substituted Quinoline with a Plk1 Inhibitor. Molecular cancer therapeutics, 20(10), 1893-1903.
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4

Antibody and Protein Assay Protocol

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Anti-MSI2 (#ab76148), anti-MSI1 (#ab21628), and anti-β-actin HRP conjugated (#ab49900) antibodies and recombinant MSI2 protein (#ab167853) were obtained from Abcam, (Cambridge, UK). Anti-EGF receptor (#4267), phospho-EGFR (Y1068) (mAb #2234), HER3/ErbB3 (#12708), HER2/ErbB2 (#4290), Smad3 (#9523), phospho-AKT (T308) (#13038), total AKT (#2920), phospho-ERK (T202/Y204) (#4370), total ERK (#4696), phospho-p70S6K (T389) (#9234), total p70S6K (#2708), and normal Rabbit IgG (#2729) were obtained from Cell Signaling, (Danvers, MA). Erlotinib and afatinib were obtained from LC Laboratories (Woburn, MA), doxycycline from Sigma-Aldrich (D9891, Darmstadt, Germany). SUPERase-In RNAse inhibitor was obtained from Thermo Fisher Scientific, (AM2694 Waltham, MA). Osimertinib was obtained from Selleckchem (#S7297, Houston, TX)
Makhov P., Bychkov I., Faezov B., Deneka A., Kudinov A., Nicolas E., Brebion R., Avril E., Cai K.Q., Kharin L.V., Voloshin M., Frantsiyants E., Karnaukhov N., Kit O.I., Topchu I., Fazliyeva R., Nikonova A.S., Serebriiskii I.G., Borghaei H., Edelman M., Dulaimi E., Golemis E.A, & Boumber Y. (2021). Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC). Oncogenesis, 10(3), 29.
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5

Western Blot Analysis of Liver Proteins

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Proteins from total liver or cellular lysates or immunoprecipitated were separated by SDS-PAGE, and probed with different primary antibodies as specified in each figure legend. The specific signals were amplified by addition of horseradish peroxidase-conjugated secondary antibodies and visualized with enhanced chemiluminescence (ECL from Millipore, MA, USA). Western blotting images were processed using a ChemiDoc XRS digital imaging system with Quantity One 1-D analysis software (Bio-Rad Laboratories, Inc., Hercules, CA, USA)
Antibodies from Cell Signaling Technology (working dilution 1:1000) were the following: phospho-Akt (Ser473) (#4060), phospho-Akt (Ser308) (#13038), phospho-Akt2 (#8599) phospho-GSK3 (Ser9)(#9327), phospho-FoxO1-3 (Thr24/32) (#9464), phospho-p70S6K (Thr389) (#9234), phospho-Glycogen Synthase (Ser641) (#3891), total Akt1 (#2967), total Akt2 (#3063, #5239), total Glycogen Synthase (#3893), total FoxO3 (#12829), total GSK3 (#12456), total p70S6K (#2708), APPL1(#3858), Rab5(#2143) (#3547), Rab7(#2094), Myc-Tag (#2272). Antibody to Glut2 (working dilution 1:1000) was from Santa Cruz biotechnology Inc (sc-9117). Original gel images are shown in Supplementary Fig. 9.
Braccini L., Ciraolo E., Campa C.C., Perino A., Longo D.L., Tibolla G., Pregnolato M., Cao Y., Tassone B., Damilano F., Laffargue M., Calautti E., Falasca M., Norata G.D., Backer J.M, & Hirsch E. (2015). PI3K-C2γ is a Rab5 effector selectively controlling endosomal Akt2 activation downstream of insulin signalling. Nature Communications, 6, 7400.
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6

Selinexor Treatment for Cancer Investigations

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selinexor (KPT-330) and Pluronic F-68 were obtained from Karyopharm Therapeutics (Newton, MA, USA). For in vitro administration, selinexor was dissolved in dimethyl sulfoxide (Sigma-Aldrich) to a concentration of 20 mM/L. For in vivo administration, selinexor was dissolved in 0.6% w/v aqueous Pluronic F-68 (Karyopharm Therapeutics, Newton, MA, USA). Antibodies to XPO1/CRM1 (H300), C-Myc, p27 (C-19), p53 (DO-1), Bax (N20), cyclin D1 (A-12) were from Santa Cruz Biotechnologies (Dallas, TX, USA). Antibodies against p21, MCL1, cleaved PARP, cleaved caspase-9, cleaved caspase-3, Aurora-B, AXL, total AKT, p-AKT, total P70S6K, p-P70S6K and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against β-actin was from Sigma-Aldrich.
Garg M., Kanojia D., Mayakonda A., Ganesan T.S., Sadhanandhan B., Suresh S., S. S., Nagare R.P., Said J.W., Doan N.B., Ding L.W., Baloglu E., Shacham S., Kauffman M, & Koeffler H.P. (2017). Selinexor (KPT-330) has antitumor activity against anaplastic thyroid carcinoma in vitro and in vivo and enhances sensitivity to doxorubicin. Scientific Reports, 7, 9749.
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7

Western Blot Analysis of Phosphorylated Proteins

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Cells and homogenized tissues were lysed in radioimmunoprecipitation assay buffer supplemented with Complete Protease Inhibitors and PhosSTOP (Roche) and proteins were processed for western blot analyses as described (28 (link)). Immunoblots were probed with antibodies that recognize phosphorylated (Ser473)-AKT, total AKT, phosphorylated (Thr24/32) FoxO1/3a, phosphorylated (Tyr-416)-Src, total Src, phosphorylated (Thr389) p70S6K, total p70S6K, phosphorylated (Ser235/236)-S6, total S6 (all from Cell Signaling) and β-actin (Sigma-Aldrich). Densitometry was performed using ImageJ (NIH, rsbweb.nih.gov/ij/).
Yori J.L., Lozada K.L., Seachrist D.D., Mosley J.D., Abdul-Karim F.W., Booth C.N., Flask C.A, & Keri R.A. (2014). Combined SFK/mTOR inhibition prevents rapamycin-induced feedback activation of AKT and elicits efficient tumor regression. Cancer research, 74(17), 4762-4771.
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8

Comprehensive Protein Analysis Protocol

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SDS-PAGE and western blotting was carried out using standard methods. The following antibodies were used: PIM1 (12H8, Santa Cruz), PIM1 (A300–313A, Bethyl Laboratories), Actin (ab6276, Abcam), GAPDH (G8795, Sigma), SUMO2 (51–9100, Zymed), His-tag (27-4710-01, GE Healthcare), HA-tag (12CA5, Sigma), Flag-tag (F1804, Sigma), MYC-tag (9E10, Hybridoma supernatant), GFP-tag (sc-8334, Santa Cruz), GST-tag (sc-459, Santa Cruz), total ERK1/2 (ER16, Transduction lab), and phospho S10 Histone H3 (06–570, Millipore). The following antibodies were purchased from Cell Signaling Technology: phospho S62 c-MYC (13748), total c-MYC (5605), total Histone H3 (4499), phospho S112 Bad (5284), total Bad (9239), phospho T37/46 4E-BP1 (2855), total 4E-BP1 (9644), phospho T389 p70S6K (108D2), total p70S6K (49D7), phospho S473 AKT (D9E), total AKT (40D4), phospho T202/Y204 ERK1/2 (9106) and phospho tyrosine-100 (9411). Sheep polyclonal UBC9 and chicken polyclonal RNF4 were from Ron Hay, University of Dundee. Secondary antibodies were purchased from Biorad and Thermo Fisher Scientific.
Iyer R.S., Chatham L., Sleigh R, & Meek D.W. (2017). A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity. Scientific Reports, 7, 3598.
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9

Signaling Pathway Antibody Analysis

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Antibodies against the following proteins were purchased from Cell Signaling Technology (USA): phosphorylated (p‐) AMPKα (Thr172) (used in experiments at 1:1,000 dilution), total AMPKα (at 1:1,000 dilution), p‐ACC (Ser79) (at 1:1,000 dilution), total ACC (at 1:1,000 dilution), p‐ERK1/2 (Thr202/Tyr204) (at 1:2,000 dilution), total ERK1/2 (at 1:2,000 dilution), p‐p38 (Thr180/Tyr182) (at 1:1,000 dilution), total p38 (at 1:1,000 dilution), p‐JNK1/2 (Thr183/Tyr185) (at 1:1,000 dilution), total JNK1/2(at 1:1,000 dilution), p‐mTOR (Ser2448) (at 1:1,000 dilution), total mTOR (at 1:1,000 dilution), p‐p70S6K (Thr389) (at 1:1,000 dilution), total p70S6K (at 1:1,000 dilution) and HRP‐linked secondary antibody (at 1:3,000 dilution). The antibody against GAPDH (at 1:3,000 dilution) was purchased from Abclonal. In addition, the following reagents were used: thymoquinone (TQ; Sigma‐Aldrich), phenylephrine (PE; Tokyo Chemical Industry) and compound C (CpC; Selleck).
Chen H., Zhuo C., Zu A., Yuan S., Zhang H., Zhao J, & Zheng L. (2021). Thymoquinone ameliorates pressure overload‐induced cardiac hypertrophy by activating the AMPK signalling pathway. Journal of Cellular and Molecular Medicine, 26(3), 855-867.
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10

Western Blot Analysis of Cellular Proteins

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ASM cells were lysed and the proteins (10–12 µg) were resolved on a 10–12% SDS-PAGE, then transferred onto nitrocellulose membranes (Bio-Rad, USA) as previously described31 . Antibodies used were sm-α-actin (1:3000), β-actin (1:15000), α2-chain laminin (1:500), Hras (1:200), Kras (1:300), Nras (1:500) (all from Sigma, USA); phospho-ERK, total ERK, phospho-p38 MAPK, total p38 MAPK, p21 Ras, phospho-PKCα, total PKCα, phospho-PKCς, total PKCς, phospho-p70S6K, total p70S6K, cyclin D1 (all 1:1000) (all from Cell Signaling, USA); and integrin α7 (1:500) (Abcam, Cambridge, UK). Proteins were visualized on Kodak film after incubation with enhanced chemiluminescence reagents, then exposure levels were quantified by TotalLab (UK) densitometry software. Results were expressed as fold increment over Day 0 relative to β-actin.
Teoh C.M., Tan S.S., Langenbach S.Y., Wong A.H., Cheong D.H., Tam J.K., New C.S, & Tran T. (2019). Integrin α7 expression is increased in asthmatic patients and its inhibition reduces Kras protein abundance in airway smooth muscle cells. Scientific Reports, 9, 9892.
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