Anti ps6k t389

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-pS6K (T389) is a primary antibody that recognizes the phosphorylated form of ribosomal protein S6 kinase (S6K) at threonine 389. S6K is a key downstream effector of the mTOR signaling pathway and its phosphorylation at Thr389 is a marker of mTOR activity.

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Close spelling variants of this product

Rabbit anti-pS6K(T389, Anti-pS6K

4 protocols using anti ps6k t389

Evaluating hamartin-cmyc functional activity

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To evaluate the functional activity of hamartin-cmyc expressed by the AAV vector construct (AAV-CMV-hamartin-cmyc), we co-expressed HA-S6 kinase (HA-S6K), which is one of the direct substrates of mTORC1, in 293T cells (Han et al., 2012 (link)). The flag-tagged TSC1 in mammalian expression vector pcDNA3 (TSC1-FLAG) was used as a positive control. Briefly, at 20 h post-transfection, 293T cells were harvested and lysed in 0.5% NP-40 lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 50 mM NaF, 1 mM Na orthovanadate, 2 mM EDTA, and 1X Complete protease inhibitor cocktail (Roche). Cell lysates (500 μg) were then subjected to immunoprecipitation (IP) using anti-HA antibody (Covance, Berkeley, CA). Anti-HA immunoprecipitates or cell lysates were separated by 4–15% gradient SDS-PAGE (Bio-Rad, Hercules, CA) and probed with various antibodies, including anti-HA, anti-pS6K (T389) (Cell Signaling, Danvers, MA), anti-C-Myc (9E10, Developmental Studies Hybridoma Bank Iowa), or anti-FLAG (Sigma) antibodies.

Prabhakar S., Zhang X., Goto J., Han S., Lai C., Bronson R., Sena-Esteves M., Ramesh V., Stemmer-Rachamimov A., Kwiatkowski D.J, & Breakefield X.O. (2015). Survival benefit and phenotypic improvement by hamartin gene therapy in a tuberous sclerosis mouse brain model. Neurobiology of disease, 82, 22-31.

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Signaling Pathway Protein Analysis

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Cells were washed with ice-cold phosphate-buffered saline (PBS) and then solubilized with protease inhibitor (Upstate Biotechnology, Temesula, CA, USA)-containing RIPA buffer. Lysates were immunoblotted with indicated antibodies, anti-G9a, anti-DUSP4, anti-p-AMPK, anti-AMPK, anti-S6K and anti-p-S6K (T389) (Cell Signaling Technology), anti-H3K9me1 (Millipore, MA, USA), anti-GLP, anti-H3K9me2, anti-histone 3, anti-LC3B (Abcam), anti-H3K9me3, anti-caspase 3, anti-PARP, anti-p62, anti-β-actin anti-GAPDH, anti-α-tubulin (GeneTex), anti-p-Akt, anti-Akt, anti-p-ERK and anti-ERK (Santa Cruz Biotechnology, Santa Cruz, CA).

Li K.C., Hua K.T., Lin Y.S., Su C.Y., Ko J.Y., Hsiao M., Kuo M.L, & Tan C.T. (2014). Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma. Molecular Cancer, 13, 172.

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Protein Expression Analysis of Transfected Cells

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The total protein content of the transfected and control cells was extracted by mixing with RIPA lysis buffer for 30 minutes on ice. Protein concentration was determined using a BCA Quantification Kit (Beyotime, Beijing, China). Protein lysates (50 μg) were separated by 12% SDS‐PAGE and blotted onto a 0.45‐μm PVDF membrane (Millipore, Hercules, CA, USA). The membrane was then incubated with HRP‐conjugated secondary antibody (Jackson, USA) at room temperature for 2 hours. Immuno‐reactivity of the separated proteins was visualized using the ECL Western Blotting kit (Invitrogen, USA) according to the manufacturer's instructions, using GAPDH as a loading control. Rabbit anti‐AKT, anti‐pAKT‐S473, anti‐S6K and anti‐pS6K‐T389 were purchased from Cell Signaling Technology (Boston, MA, USA). Anti‐rabbit IgG‐HRP and GAPDH mRB antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All experiments were performed in triplicate.

Liu J., Bian T., Feng J., Qian L., Zhang J., Jiang D., Zhang Q., Li X., Liu Y, & Shi J. (2017). miR‐335 inhibited cell proliferation of lung cancer cells by target Tra2β. Cancer Science, 109(2), 289-296.

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Antibody Reagents for Signaling Pathways

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Anti-Akt (#4685), anti-phospho-Akt S473 (#4060), anti-phospho-Akt T308 (#9275), anti-Cyclin E (#4129), anti-GSK3β (#9315), anti-phospho-GSK3β (#9336), anti-NDRG1 (# 5196), anti-phospho-NDRG1 (#3217), anti-p110α (#4249), anti-S6K (#2705), anti-pS6K T389 (#9205), anti-SGK3 (#8156), anti-Vps34 (#4263) and anti-β-actin (#4970) were purchased from Cell Signaling Technologies. Anti-FLAG M2 (#F3165) was from Sigma-Aldrich. anti-SGK3 pT320 (# S1010-85W8) was from US Biologicals. Anti-GFP (# sc-9996), anti-GST (# sc-459) and anti-INPP4B (#sc-12318) were from Santa Cruz. Anti-Fbw7 (#A301–720A) was from Bethyl Laboratories. anti-SGK3 (#LS-C132061) for immunoprecipitation was from LifeSpan BioSciences. Anti-HA was generated and purified from the 12CA5 hybridoma. Anti-p85, generated in house, has been described. Horseradish peroxidase-conjugated anti-goat was from Millipore. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulin G antibodies were from Chemicon.

Gasser J.A., Inuzuka H., Lau A.W., Wei W., Beroukhim R, & Toker A. (2014). SGK3 mediates INPP4B-dependent PI 3-Kinase signaling in breast cancer. Molecular cell, 56(4), 595-607.

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