Anti phospho p70s6k t389

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-phospho-p70S6K-T389 is a specific antibody that detects the phosphorylation of p70 S6 kinase at threonine 389. This phosphorylation site is important for the activation of p70 S6 kinase, which plays a key role in the regulation of protein synthesis and cell growth.

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P70S6K (353 citations) Anti-p70S6K (114 citations) Phospho-p70S6K (103 citations) Anti-phospho-p70S6K (53 citations) Phospho-p70S6K (T389) (15 citations) Anti-p70S6K-T389 (2 citations)

5 protocols using anti phospho p70s6k t389

Apoptosis and Mitochondrial Assays

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7-AAD and Annexin-V-FITC were from Cayman Chemical (Ann Arbor, MI, USA). Mouse IFN-γ was from eBiosciences (San Diego, CA, USA). LPS and oligomycin were from Sigma Aldrich (St. Louis, MO, USA). JC-1, MitoSOX and CellROX Green were from Molecular Probes, Life Technologies Corporations (Grand Island, NY, USA). Rabbit polyclonal anti-phospho-p70S6K-T389, laminin, HIF-1alpha, phospho-Akt-S473 and p70S6K were from Cell Signaling Technologies (Danvers, MA, USA). Goat polyclonal anti-Akt and rabbit polyclonal anti-Actin were from Santacruz Biotechnologies (Dallas, TX, USA). Goat anti-Rabbit IgG-IRDye 800CW and Donkey anti-Goat IgG-IRDye 800CW were from LI-COR BioSciences (Lincoln, NE, USA).

Matta S.K, & Kumar D. (2016). Hypoxia and classical activation limits Mycobacterium tuberculosis survival by Akt-dependent glycolytic shift in macrophages. Cell Death Discovery, 2, 16022-.

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Kahweol Acetate Signaling Pathway Analysis

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Kahweol acetate was purchased from LKT laboratories Inc. (St. Paul, MN, USA, 81760-47-6). A 40 mM solution of kahweol was initially prepared in DMSO, stored as small aliquots at −20 °C, and thawed and diluted in cell culture medium, as required. The anti-cleaved-caspase 3 (9664S,1:2000), anti-PARP (9542S,1:2000), anti-phospho-Src(2101S,Y416), anti-phospho-mTOR (S2448) (2971S1:1000), anti-phospho-p70S6K(T389) (9206S,1:1000), anti-phospho-4EBP1(T37/46) (2855S,1:5000), anti-phospho-Akt (S473) (9271S,1:2000), anti-phospho-STAT3(Y705) (9138S,1:4000), beta-tubulin (2146S,1:5000), anti-GAPDH (2118S,1:5000), anti-rabbit (7074P2,1:5000~1:10,000), and anti-mouse (7076P2,1:5000~1:10,000) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Seo H.Y., Lee S.H., Lee J.H., Lee J.H., Jang B.K, & Kim M.K. (2021). Kahweol Induces Apoptosis in Hepatocellular Carcinoma Cells by Inhibiting the Src/mTOR/STAT3 Signaling Pathway. International Journal of Molecular Sciences, 22(19), 10509.

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Palmitic Acid Signaling Pathway Modulation

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Palmitic acid (C16:0, PA) (Sigma, Burlington, MA, USA) was added to the cell culture medium as a PA-BSA complex, as described[8 (link)]. mTOR inhibitor RAD001 (20 nM, 16h) and S6K inhibitor NaSal (10 mM, 16h) were purchased from MedChemExpress (Monmouth Junction, NJ, USA) and Selleck (Houston, TX, USA), respectively. The proteasome inhibitor MG132 (20 μM, 6h) was purchased from A.G Scientific (San Diego, CA, USA). The lysosomal inhibitor leupeptin (50 μM, 16h) was purchased from Sigma (Burlington, MA, USA). The GSK3β inhibitor TWS119 (10 μM, 16h) was obtained from Santa Cruze (Dallas, Texas, USA). Cycloheximide (50 μM) was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PTEN, anti-Phospho-PTEN (Ser380), anti-p70S6K, anti-Phospho-p70S6K (T389) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Phospho-PTEN (Thr366) antibody was purchased from Abcam (Cambridge, MA, USA). Anti-vinculin, anti-HA, anti-WWP2 antibodies and PTEN-siRNA (EHU106441) were purchased from Sigma (Burlington, MA, USA). WWP2-siRNA (sc-40362) and control-siRNA (sc-37007) were obtained from Santa Cruze (Dallas, Texas, USA). S6K and mTOR inhibitors were added to cells for 1 h before PA treatment.

Massuda E.S., Dunphy E.J., Redman R.A., Schreiber J.J., Nauta L.E., Barr F.G., Maxwell I.H, & Cripe T.P. (1997). Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells. Proceedings of the National Academy of Sciences of the United States of America, 94(26), 14701-14706.

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Molecular Signaling Pathway Analysis

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The following antibodies were used for Western blot analyses, immunoprecipitation, and immunohistochemistry: anti-mTOR, anti-phospho-mTOR^Thr2448, anti-p70S6K, anti-phospho-p70S6K^T389, anti-4EBP1, anti-phospho-4EBP1^Thr37/46, anti-phospho-4EBP1^Ser65 and anti-phospho-HSP27 from Cell Signaling; anti-PRAK, anti-HIF-1α and anti-MMP2 from Proteintech (Chicago, IL), anti-tubulin and anti-GAPDH from Ruiying Biological (Suzhou, China), anti-vimentin from Gene Tex (San Antonio, Texas), anti-Snail1, anti-MMP9, anti-CDH1(E-cadherin) and anti-CDH2 (N-cadherin) from ABclonal (Wuhan, China), anti-Twist1 from Signalway Antibody (College Park, Maryland), Isotype control IgG from Sigma. PRAK inhibitor GLPG0259 was synthesized by Wuxi Pharma (Shanghai, China). MK2 inhibitor PF 3644022 was purchased from R&D systems. mTORC1 inhibitor rapamycin and mTORC1 agonist MHY1485 were from Selleck (Houston, TX), Texas red-labeled tomato lectin was from Vector Laboratories (Burlingame, CA).

Wang Y., Wang W., Wu H., Zhou Y., Qin X., Wang Y., Wu J., Sun X.Y., Yang Y., Xu H., Qian X., Pang X., Li Y., Zhang Z., Han J, & Zhang Y. (2021). The essential role of PRAK in tumor metastasis and its therapeutic potential. Nature Communications, 12, 1736.

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Immunoblotting analysis of autophagy and mTOR signaling

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The cells were collected and lysed in a cell lysis buffer (50 mM Tris–HCl (pH 7.6) containing protease inhibitor cocktail (Roche, 4693132001), 0.5% sodium deoxycholate, 1% NP-40, and 150 mM NaCl). The proteins were separated by 13% polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred onto a polyvinylidene difluoride membrane (PVDF membrane; Millipore, IPVH00010). The following primary and secondary antibodies were used: anti-GAPDH (Proteintech, 60,004-4-lg; 1:8000), anti-LC3 (Novus Biologicals, NB1-02200; 1:1000), anti-p62 (Enzo Life, BML-PW9860; 1:500), anti-phospho-p70S6K (T389) (Cell Signaling Technology, 9205; 1:100), anti-p70S6K (Epitomics, 1494–1; 1:1000), anti-TFEB (Cell Signaling Technology, 4240; 1:1000), anti-phospho-TFEB (S211) (Cell Signaling Technology, 37,681; 1:100), and RagB antibodies (Santa Cruz Biotechnology, sc-293349; 1:100) over night at 4°C; and horseradish peroxidase conjugated sheep anti-mouse and anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) for 2 h at room temperature. The proteins were visualized using an ECL detection kit (Vazyme, E411-04).

Li N., Rao L., Zhao X., Shen J., Su D., Ma G., Sun S., Ma Q., Zhang L., Dong C., Tam K.Y., Prehn J.H., Wang H, & Ying Z. (2023). Chlorpromazine affects autophagy in association with altered Rag GTPase–mTORC1–TFEB signaling. Frontiers in Cell and Developmental Biology, 11, 1266198.

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