Phospho s6k

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-S6K is a laboratory reagent that detects the phosphorylation of the S6 protein kinase, a key regulator of protein synthesis and cell growth. It is a useful tool for monitoring the activity of the mTOR signaling pathway.

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Lab products found in correlation

35 protocols using phospho s6k

Western Blot Analysis of Inhibitor-Treated Cells

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Western blot analysis of the inhibitor-treated cells was performed using a standardized protocol [35 (link)]. The primary human antibodies used in these analyses included c-MYC, SMO, and β-Actin (Santacruz, CA), AKT, phospho-AKT, S6K, phospho-S6K, GLI1 and SOX2 (Cell Signaling Technology, MA) and, cyclin D1, Bcl-2 and CD133 (BD Biosciences, CA). Immunoreactivity was detected using appropriate peroxidase-conjugated secondary antibodies (Santacruz, CA) and visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, IL).

Chaturvedi N.K., Kling M.J., Coulter D.W., McGuire T.R., Ray S., Kesherwani V., Joshi S.S, & Sharp J.G. (2018). Improved therapy for medulloblastoma: targeting hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy. Oncotarget, 9(24), 16619-16633.

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Artocarpin Modulates Cell Signaling Pathways

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The human cutaneous SCC cell line HSC-1 was obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan) and was cultured in Dulbecco's Modified Eagle's Medium with 20% FBS. Cells were incubated at 37°C with humidified air containing 5% CO₂.
Artocarpin was purchased from Pulin Biotech Company Limited (Taipei, Taiwan). The purity of artocarpin was determined to be greater than 98% by high performance liquid chromatography (HPLC) analysis. Phospho-ERK, phospho-p38, and phospho-S6K antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); phospho-JNK antibody was purchased from Santa Cruz Biotechnology (Dallas, TX); phospho-Akt antibody was from Millipore (Billerica, MA, USA); phospho-mTOR and GAPDH antibodies were from Epitomics (Burlingame, CA, USA).

Hu S.C., Lin C.L., Cheng H.M., Chen G.S., Lee C.W, & Yen F.L. (2015). Artocarpin Induces Apoptosis in Human Cutaneous Squamous Cell Carcinoma HSC-1 Cells and Its Cytotoxic Activity Is Dependent on Protein-Nutrient Concentration. Evidence-based Complementary and Alternative Medicine : eCAM, 2015, 236159.

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Immunoblot Analysis of Cell Signaling Pathways

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CD8⁺ T cells were lysed with RIPA buffer (50mM Tris-HCl, pH 8.0, 1% NP-40, 0.5% Sodium deoxycholate, and 150mM NaCl) with addition of Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and protein concentration was determined using the Bio-Rad protein assay (Hercules, California, U.S.A.). Proteins were resuspended in LDS Sample Buffer, loaded (15 μg/lane) onto NuPAGE Bis-Tris gels, and transferred to polyvinylidene difluoride (PVDF) membranes using iBlot2 system (LifeTechnologies, Grand Island, New York, U.S.A.). Proteins were detected using rabbit anti-S6K, phospho-S6K, AMPKα, phospho-AMPKα, Acetyl-CoA carboxylase (ACC), phospho-ACC, cyclin E, cdk2, phospho-Rb (Ser780), tubulin, and anti-rabbit IgG HRP-linked antibody from Cell Signaling Technology (Danvers, Massachusetts, U.S.A.). Primary antibodies were used at a 1:1,000 dilution whereas secondary antibody was used at a 1:50,000 dilution. PVDF membranes were developed using ECL Western Blotting Substrate (Thermo Scientific, Waltham, Massachusetts, U.S.A.) and exposed to Amersham hyperfilm ECL (GE Healthcare Life Sciences, Piscataway, New Jersey, U.S.A.)

Lee P.H., Yamada T., Park C.S., Shen Y., Puppi M, & Lacorazza H.D. (2015). G0S2 modulates homeostatic proliferation of naïve CD8+ T cells and inhibits oxidative phosphorylation in mitochondria. Immunology and cell biology, 93(7), 605-615.

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Elaiophylin Biosynthesis from Deep-Sea Streptomyces

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Elaiophylin was prepared from the deep-sea-derived Streptomyces sp. SCSIO 1934 [44 (link)]. Rapamycin, torin1, bafilomycin A1, compound C, STO-609, and rosiglitazone were purchased from Selleck. Insulin, IBMX, and dexamethasone were purchased from Sigma-Aldrich. Antibodies recognizing phospho-AMPKα (#2535), AMPKα (# 2532), phospho-ACC (#3661), ACC (#3662), phospho-AKT (#4060), phospho-S6K (#9204), S6K (#9202), 4EBP1 (#9644), phospho-ERK (#4370), phospho-c-JUN (#3270), and LC3B (#3868) were procured from Cell Signaling. Other antibodies included anti-AKT (Proteintech, 60203-2-Ig), anti-GST (Proteintech, 10000-0-AP), anti-TXNIP (Abcam, ab188865), and anti-ACTIN (Genescript, A00702).

Bao R., Meng Y., Zhang H., Yang C., Li W., Zhang C., Zhang J., Sun R., Li Z., Jiang W., Zhang C., Zhang C., Yuan H.X, & Dang Y. (2021). Elaiophylin reduces body weight and lowers glucose levels in obese mice by activating AMPK. Cell Death & Disease, 12(11), 972.

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Western Blot Analysis of Signaling Pathways

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Harvested cells were lysed in TBSN buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1.5 mM EDTA, 5 mM EGTA, 0.5% Nonidet P-40, and 0.5 mM Na₃VO₄) supplemented with proteinase inhibitors. The lysates were resolved by SDS-PAGE and transferred to Whatman Westran PVDF membrane (Sigma, Z671088), followed by incubation with antibodies against Plk1 (Santa Cruz, sc-17783), phospho-AKT-S473 (Cell Signaling, 9271), phospho-AKT-T308 (Cell Signaling, 13038), AKT (Cell Signaling, 9272), phospho-S6K (Cell Signaling, 9205), phospho-S6 (Cell Signaling, 2211), S6 (Cell Signaling, 2217), phospho-NFκB (Cell Signaling, 3033), NFκB (Santa Cruz, sc-372), AR (Santa Cruz, sc-7305), γ-tubulin (Sigma, T3559), p84 (Abcam, ab487), Twist1 (Sigma, SAB1411370), SREBP1 (Santa Cruz, sc-367), SREBP2 (Santa Cruz, sc-5603), phospho-GSK3β (Cell Signaling, 9322), GSK3β (BD Biosciences, 610201), FAS (BD Biosciences, 610962), HMG-CoA Reductase (EMD Millipore, ABS229), cleaved-PARP (EMD Millipore, AB3620), PSA (Cell Signaling, 5365), and β-actin (Sigma, A5441).

Zhang Z., Hou X., Shao C., Li J., Cheng J.X., Kuang S., Ahmad N., Ratliff T, & Liu X. (2014). Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer research, 74(22), 6635-6647.

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Immunohistochemical and Western Blot Analysis of Kidney Tissue

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Paraffin-embedded kidney tissue sections (5 μm) were rehydrated (2× 10 min xylene; 2× 5 min 100% EtOH; 1× 5 min 95% EtOH; 1× 5 min 70% EtOH), incubated in protease from Streptomyces griseus (Sigma, P6911; 0.75 mg/ml in Tris buffer, 0.05 M, pH 7.6) for 1 h at 37°C to retrieve antigens, blocked with Rodent Block M (BioCare Medical) for 30 min, and incubated with rabbit anti-Agtr1a antibody (Novus Bio, NBP1-77078; 1:250) in antibody diluent (Dako) for 12 h at 4°C. Western blotting on tissue lysates was performed according to standard methods, incubating the nitrocellulose membrane overnight at 4°C with the following antibodies: Agtr1a (as above; 1:750), S6K (Cell Signaling, #2708; 1:1000), phospho-S6K (Cell Signaling, #9234; 1:1000), AKT (Cell Signaling, #9272; 1:1000), and phospho-AKT^S473 (Cell Signaling, #9271; 1:1000). Quantification of p-S6K/S6K and p-AKT^S473/AKT ratios was performed as described^{46 (link), 47 (link)}.

Baker D.J., Childs B.G., Durik M., Wijers M.E., Sieben C.J., Zhong J., Saltness R., Jeganathan K.B., Versoza G.C., Pezeshki A.M., Khazaie K., Miller J.D, & van Deursen J.M. (2016). Naturally occurring p16Ink4a-positive cells shorten healthy lifespan. Nature, 530(7589), 184-189.

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Immunoblot Analysis of Cellular Proteins

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The cells were washed with PBS and lysed with radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 25 mM ethylenediaminetetraacetic acid) in 60 mm petri dishes. The collected cell lysates were stocked under refrigeration of −20°C. Equal amounts of protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (8%–12%) and transferred to a nitrocellulose membrane. The blots were incubated with primary antibodies and then probed with peroxidase-conjugated antimouse or -rabbit secondary antibodies at 4°C for 18 hours. The antibodies used were against EGFP (Abomics, Turku, Finland), p62/SQSTM1 (Abcam, Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase, Akt, phospho-Akt⁴⁷³ (Genetex, Irvine, CA, USA), S6K, phospho-S6K (Cell Signaling, Danvers, MA, USA), phospho-JNK, JNK, Beclin-1, Bcl-2 (Santa Cruz Biotechnology Inc, Dallas, TX, USA), or LC3 (MBL, Woburn, MA, USA), respectively. The immunoblots were then stained with enhanced chemiluminescence and detected by LAS3000 detector. The blot densitometries were determined using MultiGauge software (Fuji Film, Tokyo, Japan).

Hsieh C.H., Lee L.C., Leong W.Y., Yang T.C., Yao C.F, & Fang K. (2016). A triazole derivative elicits autophagic clearance of polyglutamine aggregation in neuronal cells. Drug Design, Development and Therapy, 10, 2947-2957.

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Protein Expression and Signaling Pathway Analysis

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Briefly, cells were washed with ice cold PBS and lysed with ice-cold phospholysis buffer supplemented with protease and phosphatase inhibitors. After lysis, the suspension was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected and stored for further processing. The total protein content of the extracted whole cell lysates were determined by the Bradford method, and 50μg of the total extracted protein from respective samples were denatured and subjected to immunoblotting. The following primary antibodies and their respective secondary antibodies labeled with HRP were used for detecting the expression levels of various proteins with enhanced chemiluminescence (Pierce ECL substrate, USA). Primary antibodies used were DEPTOR, eNOS, iNOS, HPV E6, HPV E7, HSC-70 (Santa Cruz, USA); mTOR, phosphor-mTOR (ser 2448), phospho-pERK1/2, phospho-p38, phospho-4EBP1, phospho-S6K, AKT, phospho-AKT, PARP, Caspase 3, Caspase 9, Caspase 7, PUMA, p53 (Cell Signaling Technology, USA); pRb (Oncogene, USA); phospho-IRS1, phospho-GSK3β (Pierce, USA); Vinculin, anti-rabbit HRP, anti-goat HRP, anti-mouse HRP antibodies (Sigma, Germany).

Srinivas K.P., Viji R., Dan V.M., Sajitha I.S., Prakash R., Rahul P.V., Santhoshkumar T.R., Lakshmi S, & Pillai M.R. (2016). DEPTOR promotes survival of cervical squamous cell carcinoma cells and its silencing induces apoptosis through downregulating PI3K/AKT and by up-regulating p38 MAP kinase. Oncotarget, 7(17), 24154-24171.

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Western Blot Analysis of Inhibitor-Treated Cells

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Western blot analysis of the inhibitor-treated cells was performed using a standardized protocol [22 (link)]. The primary human antibodies used in these analyses included NF-κB (p65), Smo, p53, p21, Nestin and β-actin (Santacruz, CA), IκBα, phospho-IκBα, phospho-NF-κB (p65), Bcl-2, S6K, phospho-S6K, 4E-BP1, phospho-4E-BP1, PLK1 and N-Myc (Cell Signaling Technology, MA) and cyclin D1, Bcl-2, and CD133 (BD Biosciences, CA). Immunoreactivity was detected using appropriate peroxidase-conjugated secondary antibodies (Santacruz, CA) and visualized using an enhanced chemiluminescence (ECL) detection system (Pierce, IL).

Chaturvedi N.K., McGuire T.R., Coulter D.W., Shukla A., McIntyre E.M., Sharp J.G, & Joshi S.S. (2016). Improved therapy for neuroblastoma using a combination approach: superior efficacy with vismodegib and topotecan. Oncotarget, 7(12), 15215-15229.

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Western Blot Analysis of Signaling Proteins

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Total cell lysates were obtained as described before [14 (link)] and analyzed by SDS-PAGE using primary antibodies against ABL1 (Santa Cruz Biotechnology, sc-56887), phospho-ABL1 (Cell Signaling, 2865), AML1 (Cell Signaling, 4334), NUP98 (Santa Cruz Biotechnology, sc-101546), phospho-CHK1 (Cell Signaling, 2348), CHK1 (Cell Signaling 2360), phospho-CHK2 (Abcam, ab59408), CHK2 (Cell Signaling, 2662), phospho-S6K (Cell Signaling, 9204), S6K (Cell Signaling, 9202), phospho-AKT (Cell Signaling, 9271), AKT (Cell Signaling, 2920), γ-H2AX (Cell Signaling, 2577), histone H3 (Invitrogen, AHO1432) and β-actin (Santa Cruz Biotechnology, sc-47778). The following secondary antibodies conjugated to HRP were used: goat anti-mouse IgG (EMD Millipore, 12-349) and goat anti-rabbit IgG (EMD Millipore, 12-348).

Golovine K., Abalakov G., Lian Z., Chatla S., Karami A., Chitrala K.N., Madzo J., Nieborowska-Skorska M., Huang J, & Skorski T. (2023). ABL1 kinase as a tumor suppressor in AML1-ETO and NUP98-PMX1 leukemias. Blood Cancer Journal, 13(1), 42.

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