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Phospho p70s6k1

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Phospho-p70S6K1 is a primary antibody that specifically recognizes the phosphorylated form of the p70S6K1 protein. p70S6K1 is a serine/threonine kinase that plays a key role in the regulation of protein synthesis and cell growth. This antibody can be used to detect the activation status of p70S6K1 in various cell and tissue samples.

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6 protocols using phospho p70s6k1

1

Quantifying Liver Protein Levels in Tg(TXN2) Mice

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Levels of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) were measured in the total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice by Western blot analysis using mouse p70S6K1, phospho-p70S6K1, 4E-BP1, and phospho-4EBP1 antibodies (Cell Signaling Technology, Inc., Danvers, MA).
Total cell lysates from liver of young (4-6 months old)Tg(TXN2)+/0 and WT mice were prepared, and detection of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was performed using Western blots. The amount of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
Total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice were prepared, and detection of HIF-1α (Catalog No. Ab82832; Abcam, Cambridge, MA) was performed using Western blots. The amount of HIF-1α was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
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2

Salternamide A Biochemical Assay

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Salternamide A (SA, Figure 1A) was dissolved in 100% DMSO and stored at −20 °C for subsequent analysis. Cobalt (II) chloride (CoCl2) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for HIF-1α, Akt, phospho-Akt (Thr308), PI3K, phospho-PI3K (Tyr458/199), RPS6, phospho-RPS6 (Ser235/236), p70S6K1, phospho-p70S6K1 (Thr389), phospho-STAT3 (Tyr705), mTOR, phospho-mTOR (Ser2448), 4E-BP1, phospho-4E-BP1 (Thr37/46), eIF4E, phospho-eIF4E (Ser209), phospho-CDC2 (Thr161), CDC25C, phospho-CDC25C (Ser216), Chk1, phospho-Chk1 (Ser345), phospho-Chk2 (Thr168), caspase-3, caspase-8, caspase-9, cleaved caspase-3, cleaved caspase-8, and LC3B were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for ERK 1/2, phospho-ERK 1/2 (Thr202/Tyr204), STAT3, CDC2, cyclin B1, cyclin A, Bcl-2, and Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for VHL, PARP, cleaved PARP, and Bim were purchased from BD Pharmingen™ (BD Biosciences, San Jose, CA, USA). Hsp90 antibody was purchased from Stressgen Bioreagents (Ann Arbor, MI, USA).
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3

Quantifying Liver Protein Levels in Tg(TXN2) Mice

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Levels of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) were measured in the total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice by Western blot analysis using mouse p70S6K1, phospho-p70S6K1, 4E-BP1, and phospho-4EBP1 antibodies (Cell Signaling Technology, Inc., Danvers, MA).
Total cell lysates from liver of young (4-6 months old)Tg(TXN2)+/0 and WT mice were prepared, and detection of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was performed using Western blots. The amount of p70S6K1 and 4E-BP1 (phosphorylated and non-phosphorylated forms) was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
Total cell lysates from liver of young (4-6 months old) Tg(TXN2)+/0 and WT mice were prepared, and detection of HIF-1α (Catalog No. Ab82832; Abcam, Cambridge, MA) was performed using Western blots. The amount of HIF-1α was quantified by a densitometer, and the data were expressed as the relative amount of protein in lysates using β-actin as an internal standard.
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4

Muscle Tissue Homogenization and Protein Analysis

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Approximately 30 to 40 mg of muscle tissue was minced in 10 μL/mg of homogenization buffer (50 mM Tris-HCL, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 50 mM sodium fluoride, and one protease inhibitor tablet per 10 mL of homogenization buffer, pH 7.5) on ice using small scissors and a Teflon pestle before being shaken for 10 minutes at 1500 rpm at room temperature and centrifuged at 11,000 rpm at 4°C for 5 minutes. The supernatant containing the sarcoplasmic proteins was transferred to a 2-mL eppendorf for western blotting according to our previous work (35 (link)). The remaining myofibrillar pellet was processed and analyzed for 13C6 enrichment according to our previous work (35 (link)). Primary antibodies used for western blotting were: phospho p70S6K1 Thr389 (#9205), total p70S6K1 (#9202), phospho–eukaryotic initiation factor 4E binding protein (4E-BP1) Thr37/46 (#9459), total 4E-BP1 (#9452), phospho–eukaryotic elongation factor 2 (p-eEF2) Thr56 (#2331), total eEF2 (#2332), phospho–protein kinase B (Akt) Ser473 (#3787), and total Akt (#9272) from Cell Signaling Technology (New England Biolabs Ltd, Hitchin, United Kingdom).
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5

Glioma Cell Line Characterization

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Human glioma cell lines U87 and U251 (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 2 mmol/L l-glutamine, 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin, 5% CO2 at 37°C. Antibodies against GSK-3β, phospho-GSK-3β (Ser9), β-catenin, AKT, phospho-AKT (Ser473), p70S6K1, phospho-p70S6K1, mTOR, phospho-mTOR, β-Tubulin were obtained from Cell Signaling (Beverly, MA, USA). HIF-1α was from BD Biosciences (Franklin Lakes, NJ, USA), and GAPDH was purchased from KangCheng Biotech (Shanghai, China), while antibody against CD31 was supplied by Abcam (Cambridge, UK).
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6

Western Blot Analysis of Autophagy Markers

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Six hundred thousand cells/well were plated in 6-well plates, 24 hours prior drug treatment. Following drug treatments whole cell lysates were prepared of adherent and floating cells with RIPA lysis buffer containing 1X EDTA-free protease inhibitor cocktail (Roche) and 2X PhosphoSTOP (Roche). Dosed protein samples were separated on pre-cast 4–12 % Bis-Tris gels (Invitrogen) and transferred to nitrocellulose using the iBlot dry blotting system (Invitrogen). Blocked membranes were incubated with primary antibodies against TFEB (#101532; Santa Cruz), LAMP1 (# H4A3-s; Hybridoma Bank), LC3 (#2775; Cell Signaling), 4E-BP1 (#9452; Cell Signaling), phospho-4E-BP1 (#9459S; Cell Signaling), p70-S6K1 rabbit IgG (#9202S; Cell Signaling), phospho-p70-S6K1 (#9205S; Cell Signaling) and GAPDH (#25778; Santa Cruz). HRP-conjugated anti-rabbit IgG (#213110-01; GeneTex) and anti-mouse IgG (#213111-01; GeneTex) antibodies were used as secondary antibodies. For immunodetection membranes were incubated with peroxide and luminol solution (1:1) and analyzed with a chemiluminescence imager (Intas). Protein bands were quantified using the gel analysis tool of ImageJ and normalized to the loading control GAPDH. Blots shown are representative of at least three independent experiments.
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