Phospho p70s6k

Manufactured by Santa Cruz Biotechnology

Phospho-p70S6K is a laboratory reagent used to detect and measure the phosphorylation level of the p70S6 kinase protein, which is a key regulator of cell growth and proliferation. This product provides a reliable and specific tool for researchers to study the activation state of this important signaling pathway.

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5 protocols using phospho p70s6k

Isolation and Characterization of HyFS

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3′-epi-12β-hydroxyfroside (HyFS) was isolated and purified (> 95%) by our collaborating group 5 . Other reagents used in this study were purchased from Chinese suppliers indicated in the brackets. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma, M2128), dimethylsulfoxide (DMSO, Sigma, D2650), Z-VAD-fmk (Sigma, V116), wortmannin (Sigma, W1628), bafilomycin A1 (Sigma, B1793), CQ (Sigma, C6628), 3-MA (MCE, HY-13259), MK-2206 (MCE, HY-10358), 17-AAG (MCE, HY-10211), MG-132 (Sigma, M7449), CHX (MCE, HY-12320). The following antibodies were also purchased from Chinese suppliers indicated in the brackets. β-actin (Santa Cruz, sc-1616), caspase 3 (Millipore, MAB4703), PARP (Millipore, 04-575), LC3B (MBL, PM036), BECLIN1 (Santa Cruz, sc-11427), ATG5 (Cell Signaling Technology, 2630), SQSTM1 (MBL, M162-3), Akt (Cell Signaling, 4685), phospho-Akt (Cell Signaling, 4051), p70S6K (Santa Cruz, sc-9027), phosphop70S6K (Santa Cruz, sc-7984-R), mTOR (Millipore, 04-385), p-mTOR (Millipore, 09-213), Hsp90 (Santa Cruz, sc-27987), HA-tag (Santa Cruz, sc-53516).

Sun Y., Huang Y.H., Huang F.Y., Mei W.L., Liu Q., Wang C.C., Lin Y.Y., Huang C., Li Y.N., Dai H.F, & Tan G.H. (2018). 3′-epi-12β-hydroxyfroside, a new cardenolide, induces cytoprotective autophagy via blocking the Hsp90/Akt/mTOR axis in lung cancer cells. Theranostics, 8(7), 2044-2060.

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Western Blot Analysis of Kidney Proteins

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Protein from the kidney cortex or HK-2 cells was extracted and western blot analysis was performed as described previously47 (link). Antibodies used in this study included: collagen I, collagen IV (Southern Biotech), CTGF, Phospho-p70s6k, p70s6k, β-actin (Santa Cruz), CD32b (R&D), Phospho- NF-κB/p65, NF-κB/p65, phospho-Smad3, Smad3, Phospho-mTOR (Ser2448), mTOR, Phospho-ERK1/2 MAPK, ERK1/2 MAPK, Phospho-p38 MAPK, p38 MAPK (Cell Signaling), and LI-COR IRDye 800-labelled secondary antibodies (Rockland Immuno-chemicals). The detection of specific signals was performed by using the Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE, USA) and quantified by Image J software (National Institutes of Health). The ratio for the protein detected was normalized against β-actin and expressed as the mean ± S.E.M.

You Y.K., Huang X.R., Chen H.Y., Lyu X.F., Liu H.F, & Lan H.Y. (2016). C-Reactive Protein Promotes Diabetic Kidney Disease in db/db Mice via the CD32b-Smad3-mTOR signaling Pathway. Scientific Reports, 6, 26740.

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Western Blot Analysis of Polycystic Kidney Proteins

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The following primary antibodies were used for Western blot analysis: Polycystin‐2 (sc‐10376 Santa Cruz Biotechnology), p70‐S6K (sc‐230 Santa Cruz Biotechnology), phospho‐p70‐S6K (sc‐8416 Santa Cruz Biotechnology), phospho‐mTOR (5536 CST), phospho‐4E‐BP1 (2855 CST), PTEN (9559 CST), Akt (9272 CST), phospho‐Akt (9271 CST), actin (MAB1501 Millipore), polycystin‐1 CT2741 (kindly provided by the Baltimore Polycystic Kidney Disease Research and Clinical Core Center), mTOR (701483 Thermo Fisher Scientific), 4EBP1 (AHO1382 Thermo Fisher Scientific), JAK2 (ab37226 Abcam), phospho‐JAK2 (ab32101 Abcam). The following secondary antibodies were used: goat anti‐mouse IgG HRP‐conjugate (AP124P Millipore), goat anti‐rabbit IgG HRP‐conjugate (AP132P Millipore), donkey anti‐goat IgG HRP‐conjugate (A00178 GenScript). The IgPKD1 inhibitory antibody was a generous gift from Dr O. Ibraghimov‐Beskrovnaya and H. Husson (Genzyme Co., Boston).

Papavassiliou K.A., Zoi I., Gargalionis A.N, & Koutsilieris M. (2019). Polycystin‐1 affects cancer cell behaviour and interacts with mTOR and Jak signalling pathways in cancer cell lines. Journal of Cellular and Molecular Medicine, 23(9), 6215-6227.

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Western Blot Analysis of AKT-mTOR Pathway

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An exact weight of 0.2 g brain tissue was immersed once in 0.1 M ice-cold phosphate-buffered saline (BPS). After air drying, the tissue was placed in 500 μl ice-cold RIPA lysis buffer with protease inhibitors to prepare the tissue homogenates. After 30 minutes on ice, the homogenates were centrifuged for 5 min (12,000 rpm, 4 °C), and the supernatants were collected to be used as sample solutions for western blot analysis, as described in a previous study31 (link). Adequate sample solutions were removed to measure the total protein concentrations using the bicinchoninic acid (BCA) method. The membranes were incubated with the following primary antibodies: AKT (1:2,000; Cell Signaling Technology, Boston), phospho-AKT (1:2,000; Cell Signaling Technology, Boston), mTOR (1:1,000; Cell Signaling Technology, Boston), phospho-mTOR (1:100; Cell Signaling Technology, Boston), phospho-p70S6K (1:200; Santa Cruz Biotechnology, California), p70S6K (1:1,000; Cell Signaling Technology, Boston) and β-actin (1:4,000; Santa Cruz Biotechnology, California). The band density was quantified using Image J software. The amount of protein expression is presented relative to the levels of β-actin.

Xing Z., Xia Z., Peng W., Li J., Zhang C., Fu C., Tang T., Luo J., Zou Y., Fan R., Liu W., Xiong X., Huang W., Sheng C., Gan P, & Wang Y. (2016). Xuefu Zhuyu decoction, a traditional Chinese medicine, provides neuroprotection in a rat model of traumatic brain injury via an anti-inflammatory pathway. Scientific Reports, 6, 20040.

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Comprehensive Liver Protein Analysis

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Liver samples were homogenized in RIPA buffer and then equal amounts of liver homogenates were fractionated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was incubated with antibodies to raptor, phospho-STAT3, phospho-Akt, phospho-ERK, ERK, phospho-GSK3α/β, GSK3, cyclin D1, LC3 (all from Cell signaling), phospho-p70S6K, p70S6K, cyclin B, cyclin E, cdc2, p27, STAT3 (all from Santa Cruz), tubulin (sigma), p62 (Progen), and p21 (Chemicon).

Umemura A., Park E.J., Taniguchi K., Lee J.H., Shalapour S., Valasek M.A., Aghajan M., Nakagawa H., Seki E., Hall M.N, & Karin M. (2014). Liver Damage, Inflammation and Enhanced Tumorigenesis after Persistent mTORC1 Inhibition. Cell metabolism, 20(1), 133-144.

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