P p70 s6 kinase

Manufactured by Cell Signaling Technology

Sourced in United States

P-p70 S6 kinase is a lab equipment product that measures the phosphorylation of the p70 S6 kinase protein. It is used to assess the activation status of this important cellular signaling protein.

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Lab products found in correlation

11 protocols using p p70 s6 kinase

Inhibition of AXL and EGFR Signaling

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Gefitinib (CAS No. HY-50895) and Osimertinib (AZD-9291, CAS No. HY-15772) were purchased from MedChemExpress (NJ, USA). Cycloheximide (CAS No. 66-81-9) was purchased from A.G. Scientific (CA, USA). Yuanhuadine (YD; purity >98.5%) was isolated from a CHCl₃-soluble fraction of the flowers of Daphne genkwa, as described previously^{22 (link)}. All chemicals were dissolved in DMSO for in vitro experiments.
Antibodies against C-terminal AXL (sc-1096), EGFR (sc-03), p-ERK (sc-7383), ERK (sc-94), MET (sc-10), β-actin (sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-AXL (#5724), p-EGFR (#2234), p-MET (#3077), p-Akt (#9271), Akt (#9272), p-p70S6 Kinase (#9205), p70S6 Kinase (#9202), p-SAPK/JNK (#9091), SAPK/JNK (#9252), and snail (#3879) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Kim D., Bach D.H., Fan Y.H., Luu T.T., Hong J.Y., Park H.J, & Lee S.K. (2019). AXL degradation in combination with EGFR-TKI can delay and overcome acquired resistance in human non-small cell lung cancer cells. Cell Death & Disease, 10(5), 361.

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Quantitative Western Blot Analysis of PI3K/AKT Pathway

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ASMCs were washed once with cold DPBS (#14190, Thermo Fisher Scientific) and lysed by RIPA buffer (#R0278, Sigma-Aldrich). Cell lysates were centrifuged at 12,000 rpm (10 min) and the protein concentration of the supernatant was measured by using the BCA protein assay kit (#23227, Thermo Fisher Scientific). Equal amounts of denatured proteins (20 μg) were separated in 4%–12% SDS–PAGE (#M41212, GenScript, Piscataway, NJ, USA), and, subsequently, transferred onto a nitrocellulose membrane (#88018, Themo Fisher Scientific). Proteins of interest were detected by specific antibodies, including total- (t-)PI3K (#4292), and phosphorylated- (p-)PI3K (#GTX132597), t-AKT (#4691) and p-AKT (#4060), t-p70S6 kinase (#2708) and p-p70S6 kinase (#9205), t-STAT3 (#9139) and p-STAT3 (#9145), PTEN (#9188, which were all purchased from Cell Signaling Technology, Cambridge, UK), PGC-1α (#ab54481, Abcam), t-PPARγ (#sc-7273, Santa Cruz), and p-PPARγ (#sc-28001, Santa Cruz). GAPDH (#2118, Cell Signaling Technology) was used as an endogenous control for a semi-quantification measure. Protein bands were visualized by Luminata Forte Western HRP substrate (#WBLUF, Sigma-Aldrich) after binding specie-specific secondary HRP conjugated antibodies (#7076 and #7074, Cell Signaling Technology).

Fang L., Wang X., Sun Q., Papakonstantinou E., S’ng C., Tamm M., Stolz D, & Roth M. (2019). IgE Downregulates PTEN through MicroRNA-21-5p and Stimulates Airway Smooth Muscle Cell Remodeling. International Journal of Molecular Sciences, 20(4), 875.

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RPPA-Based PI3K Pathway Characterization in TNBC PDX Models

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RPPA was performed as previously described 29 (link) for 48 of the 57 TNBC PDX (9 PDX were established after the RPPA analysis). We calculated a PI3K pathway score with normalized data to assess pathway activation. Scores were obtained by calculating the sum for positive protein components (PI3K p110 subunit β, p-AKT1 (Ser473), p-AKT1 (Thr308), p-4E-BP1, p-p70-S6 kinase, p-S6 ribosomal protein; Cell Signaling Technology®) and subtracting the negative components of the pathway (PTEN, Cell Signaling Technology®). Eleven PDX classified as unstable (UNS) in Lehmann's classification were excluded from the analysis shown in Figure 3B.

Coussy F., Lavigne M., de Koning L., Botty R.E., Nemati F., Naguez A., Bataillon G., Ouine B., Dahmani A., Montaudon E., Painsec P., Chateau-Joubert S., Laetitia F., Larcher T., Vacher S., Chemlali W., Briaux A., Melaabi S., Salomon A.V., Guinebretiere J.M., Bieche I, & Marangoni E. (2020). Response to mTOR and PI3K inhibitors in enzalutamide-resistant luminal androgen receptor triple-negative breast cancer patient-derived xenografts. Theranostics, 10(4), 1531-1543.

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Western Blot Analysis of Cellular Proteins

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Cells were lysed by scraping in boiling 1% SDS lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1% SDS), immediately boiled for 10 minutes, and frozen at -20°C. Tumors were collected in dry ice, crushed, syringed in boiling 1% SDS lysis buffer and boiled for 10 min. Protein concentrations were measured using BCA Protein Assay (Pierce, #23225). 15-30 μg of protein per condition were loaded into SDS-PAGE polyacrylamide gels and transferred onto PVDF membranes (Millipore, #IPVH00010). Membranes were blocked for 30-60 min in 5% milk in TBST buffer and incubated with primary antibodies overnight at 4°C. The following antibodies were used for detection: antibodies against Mcl-1 (#5453, #94296), Bcl-xL (#2762 and #2764), Bim (#2819), Bak (#12105), Bax (#2772), p-ERK (#4370), ERK (#9102), Vinculin (#4650), p-AKT (#4060), AKT (#9272), p-S6 ribosomal protein (#2215), S6 ribosomal protein (#2217), p-p70 S6 kinase (#9205), p70 S6 kinase (#9202), Bcl-2 (#2872) and USP9X (#14898) were purchased from Cell Signaling Technology. Antibodies against Mcl-1 (#ab32087) and total RNA Pol II (#ab817) were purchased from Abcam. Antibodies against p-S2 CTD Pol II (#04-1571), p-S5 CTD Pol II (#04-1572), and p-S7 CTD Pol II (#1570) were purchased from EMD Millipore. Antibody against alpha-Tubulin (#T5168) was purchased from Sigma.

Perurena N., Lock R., Davis R.A., Raghavan S., Pilla N.F., Ng R., Loi P., Guild C.J., Miller A.L., Sicinska E., Cleary J.M., Rubinson D.A., Wolpin B.M., Gray N.S., Santagata S., Hahn W.C., Morton J.P., Sansom O.J., Aguirre A.J, & Cichowski K. (2023). USP9X mediates an acute adaptive response to MAPK suppression in pancreatic cancer but creates multiple actionable therapeutic vulnerabilities. Cell Reports Medicine, 4(4), 101007.

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Investigating Apoptosis and Cell Signaling Pathways

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Lansoprazole and gefitinib were purchased from Selleck Chemicals (Houston, TX, United States) and Target Molecule Corp. (Boston, MA, United States), respectively. Monodansylcadaverine (MDC) and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, MO, United States). A FITC-Annexin V apoptosis detection kit was obtained from BD Bioscience (San Josè, CA, United States). RPMI 1640 and FBS were purchased from the Biological Industries (Beit Haemek, Israel). Enhanced chemiluminescence (ECL) reagent was purchased from Thermo Fisher Scientific (Waltham, MA, United States). Antibodies specific for p27, p-Rb, cyclin D1, LC3B, p-signal transducer and activator of transcription (Stat) 3, phosphatidylinositol 3-kinase (PI3K)-p110α, PI3K-p110β, p-mammalian target of rapamycin complex 1 (mTORC1) (Ser2448), Akt, p-Akt (Ser473), p-p70 S6 kinase (S6K) (Thr389), p-glycogen synthase kinase 3 (GSK-3)β, poly (ADP-ribose) polymerase (PARP), caspase-3, K-Ras, p-extracellular signal-regulated kinase (ERK)1/2, p-c-Raf, Ki67, β-actin, and horseradish peroxidase-conjugated goat anti-rabbit and horse anti-mouse secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, United States). Antibodies specific for Bcl-2 and Bax were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States).

Zhao X., Zhang N., Huang Y., Dou X., Peng X., Wang W., Zhang Z., Wang R., Qiu Y., Jin M, & Kong D. (2021). Lansoprazole Alone or in Combination With Gefitinib Shows Antitumor Activity Against Non-small Cell Lung Cancer A549 Cells in vitro and in vivo. Frontiers in Cell and Developmental Biology, 9, 655559.

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Protein Extraction and Western Blot Analysis

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Proteins were extracted in IP-MS cell lysis buffer (Life Technologies) and quantified using the Pierce BCA kit (Thermo Fisher) as previously described [21 (link)]. The following antibodies were used for immunoblotting: Vimentin (Millipore), p-P70 S6 Kinase (Cell Signaling), P70 S6 Kinase (Cell Signaling), pS6 (Cell Signaling), S6 (Cell Signaling), Cleaved-Caspase 3 (Cell Signaling), Caspase 3 (Cell Signaling), Cleaved-PARP (Cell Signaling), PARP (Cell Signaling), GAPDH (Cell Signaling), LITAF (Santa Cruz Biotechnology), and β-actin HRP (Sigma-Aldrich) as loading control. For protein expression analysis, expression was normalized to β-actin (and/or the total protein for phospho-proteins) then compared to their respective control.

Bassot A., Dragic H., Haddad S.A., Moindrot L., Odouard S., Corlazzoli F., Marinari E., Bomane A., Brassens A., Marteyn A., Hibaoui Y., Petty T.J., Chalabi-Dchar M., Larrouquere L., Zdobnov E.M., Legrand N., Tamburini J., Lincet H., Castets M., Yebra M., Migliorini D., Dutoit V., Walker P.R., Preynat-Seauve O., Dietrich P.Y, & Cosset É. (2023). Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma. Cell Death & Disease, 14(9), 630.

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Immunoblotting of Tumor Signaling Pathways

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Frozen pairs of HBV and HCV human non-tumor and tumor, and 3 primary NASH HCCs were sourced from the Westmead Liver Clinic and approved by the Westmead Hospital Human Ethics Committee. Total protein extracts from murine and human tumor tissue were subjected to immunoblotting as previously described [23 (link)]. Antibodies for AMPK (#2532), phospho-AMPK (Thr172; #2535), Akt (#9272), p-Akt (Ser 473; #4051), mTOR (#2972), p-mTOR (Ser2448; #2971, 4E-BP1 (#9644), p-4E-BP1 (Thr70 and Ser65; #9455), p70S6-kinase (#9202), p-p70S6-kinase (Thr421/Ser424; #9204), Src (#2108), p-Src (Tyr416; #2101), AFP (#3903), JNK (#9252), p-JNK (Thr183/Tyr185; #9255) and PCNA (#2586) were from Cell Signaling. Anti-β actin (A2228) antibodies were from Sigma Aldrich, and anti-albumin (NB600-41532) was from Novus Biologicals.

Walker S., Wankell M., Ho V., White R., Deo N., Devine C., Dewdney B., Bhathal P., Govaere O., Roskams T., Qiao L., George J, & Hebbard L. (2019). Targeting mTOR and Src restricts hepatocellular carcinoma growth in a novel murine liver cancer model. PLoS ONE, 14(2), e0212860.

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Protein Extraction and Western Blot Analysis

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Following treatment, cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.0% Triton X-100, 20 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF and protease inhibitors). Lysates were cleared of insoluble material by centrifugation at 10,000g for 10 min at 4°C. The cell extract protein concentrations were measured using the Bradford assay (BioRad, 500–0006) according to the manufacturer’s protocol and a BioTek plate reader. Ten micrograms of protein were separated by polyacrylamide gel electrophoresis. The following antibodies were used: TGM2 (Abcam, ab421), p-S6 (Ser 235/236, Cell Signaling #2211), p-p70 S6 Kinase (Cell Signaling #9205), β-actin (Sigma, A5441), LC3 (Sigma, L8918), and cleaved-caspase 3 (Cell signaling #9661).

Cao J, & Huang W. (2016). Compensatory Increase of Transglutaminase 2 Is Responsible for Resistance to mTOR Inhibitor Treatment. PLoS ONE, 11(2), e0149388.

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Comprehensive Protein Signaling Analysis

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The following antibodies against: PARP, PTEN, Akt, P-Akt^S473, P-ERK, ERK, P-P70 S6 kinase, mTOR, Raptor, Rictor, and IGF-1R were obtained from Cell Signaling; α-actin was from Sigma. The antibody for FAS was obtained from BD BioSciences. Negative control scrambled siRNA and siRNA targeted against mTOR, Raptor and Rictor were obtained from Dharmacon. siRNAs targeted against FAS were obtained from Santa Cruz Biotechnology. Cerulenin, LY294002 and PD0325901 were purchased from Sigma. Lipofectamine RNAiMax (Invitrogen) was used for transient transfections.

Yellen P.B, & Foster D.A. (2014). Inhibition of fatty acid synthase induces pro-survival Akt and ERK signaling in K-Ras-driven cancer cells. Cancer letters, 353(2), 258-263.

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Western Blot Analysis of Epithelial-Mesenchymal Transition Markers

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Total lysates from cells under the different experimental conditions were separated on 7.5% polyacrylamide denaturing gels and blotted onto nitrocellulose membranes (GE Healthcare Life Science, Little Chalfont, United Kingdom), which were reacted with antibodies directed against E-cadherin (sc-7870), Vimentin (sc-373717), and β-Catenin (sc-7963) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); against Akt2 (#3063), p-Akt2 (Ser474, #8599), p-GSK-3β (Ser9, #5558), GSK-3β (#9315), p-p70 S6 Kinase (Thr389, #9205), and p70 S6 Kinase (#9202) from Cell Signaling Technology (Danvers, MA, USA); and against Akt1 (#610860, BD Biosciences), anti-p-Akt1 (Ser473, #05-739), and anti-β-Tubulin (#T4026), following previously reported procedures [27 (link)]. The immunocomplexes were detected by using a WESTAR NOVA 2.0 (Cyanagen, Bologna, Italy), and the chemiluminescence-derived bands were captured with an ImageQuantTM LAS 4000 imager (GE Healthcare Life Science) and quantified with Image Quant TL software v7.0 (GE Healthcare Life Science), as previously reported [27 (link)].

Brugnoli F., Dell’Aira M., Tedeschi P., Grassilli S., Pierantoni M., Foschi R, & Bertagnolo V. (2024). Effects of Garlic on Breast Tumor Cells with a Triple Negative Phenotype: Peculiar Subtype-Dependent Down-Modulation of Akt Signaling. Cells, 13(10), 822.

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