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Phospho p70s6k t389

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Phospho-p70S6K (T389) is a primary antibody that specifically recognizes p70S6 kinase phosphorylated at threonine 389. It is used to detect and quantify the phosphorylation of p70S6 kinase, which is involved in the regulation of cell growth and proliferation.

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Lab products found in correlation

P70s6k, by Cell Signaling Technology (4 mentions) Phospho akt s473, by Cell Signaling Technology (4 mentions) Phospho ulk1 s757, by Cell Signaling Technology (3 mentions) Total akt, by Cell Signaling Technology (2 mentions) Total p70s6k, by Cell Signaling Technology (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Ampkα, by Cell Signaling Technology (2 mentions) Phospho mtor s2448, by Cell Signaling Technology (2 mentions) Phospho stat3 s727, by Cell Signaling Technology (2 mentions) Phospho mtor s2481, by Cell Signaling Technology (2 mentions) Anti rabbit alexa fluor 647, by Thermo Fisher Scientific (1 mentions) Goat anti rabbit hrp, by Thermo Fisher Scientific (1 mentions) Goat anti mouse hrp, by Thermo Fisher Scientific (1 mentions) M per extraction reagent, by Thermo Fisher Scientific (1 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions) P src y416, by Cell Signaling Technology (1 mentions) Pakt t308, by Cell Signaling Technology (1 mentions) P tyr 100, by Cell Signaling Technology (1 mentions) P tyr 1000, by Cell Signaling Technology (1 mentions) L lysine 2hcl 13c6, by Cambridge Isotopes (1 mentions)

Spelling variants of this product

P70S6K (353 citations) Phospho-p70S6K (103 citations) Anti-phospho-p70S6K-T389 (5 citations)

15 protocols using phospho p70s6k t389

1

Anti-FLAG and Phospho-Specific Antibody Protocol

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Anti-FLAG M2 (#F1804) and Anti-FLAG M2-HRP (#A8592) were purchased from Sigma-Aldrich and anti-Src antibody (#MA5-15120) was from Thermo Scientific. p-Src (Y416) (#2113), p-Abl (Y412) (#2865), p-Tyr-100 (#9411), p-Tyr-1000 (#8954), anti-GAPDH (#14C10), Myc Tag (mouse #2276 and rabbit #71D10), p-AKT (T308) (#13038), p-AKT (S473) (#4060), p70 S6 kinase (#9202), and phospho-p70 S6K (T389) (#9234) primary antibodies were purchased from Cell Signaling Technologies (CST). Secondary antibodies Goat anti-Mouse-HRP (#31430) and Goat anti-Rabbit-HRP (#31460) were from Thermo Scientific. Alexa Fluor 488 anti-mouse (#A11029) and Alexa Fluor 647 anti-rabbit (#A21245) were from Life Technologies. All primary antibodies were used at a 1:1000 dilution and secondaries at 1:5000. Protease inhibitor cocktail (#P8340), Phosphatase inhibitor cocktail 2 (#P0044) and 3 (#P5726) were purchased from Sigma-Aldrich. NeuCode labeling reagents L-Lysine:2HCl (3,3,4,4,5,5,6,6-D8, 98%) (#DLM-2641-0) and L-Lysine:2HCl (13C6, 99%, 15N2, 99%) (#CNLM-291-H-0.25) were from Cambridge Isotopes. Rapamycin was from Cayman Chemical and M-PER extraction reagent (#78501) was from Thermo Scientific. All restriction enzymes and DNA polymerases were purchased from NEB (Ipswich, MA). Oligonucleotides and gBlocks Gene Fragments were purchased from IDT and all constructs were verified by DNA sequencing (Quintara Biosciences).
Diaz J.E., Morgan C.W., Minogue C.E., Hebert A.S., Coon J.J, & Wells J.A. (2017). A Split-Abl Kinase for Direct Activation in Cells. Cell chemical biology, 24(10), 1250-1258.e4.
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2

Antibody and Protein Assay Protocol

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Anti-MSI2 (#ab76148), anti-MSI1 (#ab21628), and anti-β-actin HRP conjugated (#ab49900) antibodies and recombinant MSI2 protein (#ab167853) were obtained from Abcam, (Cambridge, UK). Anti-EGF receptor (#4267), phospho-EGFR (Y1068) (mAb #2234), HER3/ErbB3 (#12708), HER2/ErbB2 (#4290), Smad3 (#9523), phospho-AKT (T308) (#13038), total AKT (#2920), phospho-ERK (T202/Y204) (#4370), total ERK (#4696), phospho-p70S6K (T389) (#9234), total p70S6K (#2708), and normal Rabbit IgG (#2729) were obtained from Cell Signaling, (Danvers, MA). Erlotinib and afatinib were obtained from LC Laboratories (Woburn, MA), doxycycline from Sigma-Aldrich (D9891, Darmstadt, Germany). SUPERase-In RNAse inhibitor was obtained from Thermo Fisher Scientific, (AM2694 Waltham, MA). Osimertinib was obtained from Selleckchem (#S7297, Houston, TX)
Makhov P., Bychkov I., Faezov B., Deneka A., Kudinov A., Nicolas E., Brebion R., Avril E., Cai K.Q., Kharin L.V., Voloshin M., Frantsiyants E., Karnaukhov N., Kit O.I., Topchu I., Fazliyeva R., Nikonova A.S., Serebriiskii I.G., Borghaei H., Edelman M., Dulaimi E., Golemis E.A, & Boumber Y. (2021). Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC). Oncogenesis, 10(3), 29.
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3

Immunofluorescence and Western Blotting Antibodies

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Primary antibodies used for immunofluorescence microscopy and Western Blotting were obtained from Cell Signaling Technology and included: Beta-actin (#3700 and #4970), phospho-p70s6k T389 (#9234), NF kappa B p65 (#8242S), beta-tubulin (#2128S), Caspase-3 (#14220), Caspase-1 (#3866), Phospho-MLKL (#91689). Secondary antibodies used to detect the primary antibodies for Western Blotting were obtained from Thermo Fisher Scientific, and included goat anti-rabbit and goat anti-mouse IgG-HRP (#31460 and #31430). Secondary antibodies used for immunofluorescence microscopy were obtained from Molecular Probes (Life Technologies), and included goat anti-rabbit IgG AlexaFluor488 and goat anti-mouse IgG AlexaFluor594.
De-Leon-Lopez Y.S., Thompson M.E., Kean J.J, & Flaherty R.A. (2023). The PI3K-Akt pathway is a multifaceted regulator of the macrophage response to diverse group B Streptococcus isolates. Frontiers in Cellular and Infection Microbiology, 13, 1258275.
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4

Rabbit Antibody Western Blotting

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Rabbit monoclonal antibodies for western blotting against β-actin (#4970), vinculin (#4650), AMPKα (#2532), AMPKα1 (#2795), AMPKα2 (#2757), ACC1 (#3676), p70S6K (#9202), Raptor (#2280), Akt (#9272) and against phospho-AMPK [T172 (#2535) or S485 (#2537)], phospho-ACC (S79) (#3661), phospho-p70S6K (T389) (#9205), phospho-Raptor (S792) (#2083), and phospho-Akt (S473) (#9271) were obtained from Cell Signaling Technology (Frankfurt, Germany). Mouse monoclonal antibodies against the HSV-1 proteins gB (ab6506) and ICP4 (ab6514) were obtained from Abcam (Cambridge, United Kingdom).
Doshi H., Spengler K., Godbole A., Gee Y.S., Baell J., Oakhill J.S., Henke A, & Heller R. (2023). AMPK protects endothelial cells against HSV-1 replication via inhibition of mTORC1 and ACC1. Microbiology Spectrum, 11(5), e00417-23.
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5

Western Blot Analysis of mTOR Signaling

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Myocardial lysates were derived from flash frozen tissue (Cell Signaling Technology #9803) and protein concentration determined by BCA (Pierce). Samples were prepared in SDS Tris-Glycine buffer (Life Technologies) and run on TGX 7.5% or 4–15% Tris-Glycine Gels (Bio-Rad) and blotted onto a nitrocellulose membrane. The following primary antibodies were used in this study: phospho-p70 S6K (T389) #9205 lot 26 used at 1:1,000, p70 S6K #9202 lot 20 used at 1:1,000, phospho-4EBP1 (S65) #9451 lot 14 used at 1:1,000, 4EBP1 #9452 lot 12 used at 1:1,000, phospho-Ulk-1 (S757) #1420 clone D706U lot 4 used at 1:500, Ulk-1 #8054 clone D8H5 lot 5 used at 1:500, TSC2 #3612 clone D93F12 lot 6 used at 1:1,000 (Cell Signaling Technology), phospho-TSC2 (S1365) #120718 lot NFSA12072OAH used at 1:500 (NovoPro Labs), and P62 #ab109012 lot GR12843–70 used at 1:1,000 (Abcam), and a total protein stain #926–11016 lot C80522–02 used at 5 ml/membrane (Li-Cor). Antibody binding was visualized by infrared imaging (Odyssey, Li-Cor) and quantified with Li-Cor Image Studio Software 3.1.
Oeing C.U., Jun S., Mishra S., Dunkerly-Eyring B.L., Chen A., Grajeda M.I., Tahir U.A., Gerszten R.E., Paolocci N., Ranek M.J, & Kass D.A. (2021). MTORC1-Regulated Metabolism Controlled by TSC2 Limits Cardiac Reperfusion Injury. Circulation research, 128(5), 639-651.
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6

Western Blot Analysis of Cellular Signaling

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Samples containing equal amounts of protein (10 μg) were resolved by polyacrylamide gel electrophoresis (SDS-PAGE) and processed as described previously [15 (link),16 (link)]. Briefly, membranes were blocked using TBS-Tween containing 3% (w/v) BSA for 1 hour and incubated with antisera diluted in the same overnight at 4°C. Following washing in TBS-Tween, membranes were incubated with horseradish peroxidase-conjugated secondary antibody and signals developed using ECL reagent. The antiserum used were raised against: eIF4A, phospho-eIF4E, eIF4G, eIF4E [15 (link),16 (link)]; MGMT, phospho-eIF2α, (Abcam, UK); 4E-BP1, phospho-4E-BP S65, phospho-4E-BP1 T70, phospho-p70 S6K T389, phospho- ERK1/2, phospho-p38 MAPK, phospho-rpS6 S240/44, phospho-Akt T308, p21cip1 (Cell Signaling, UK).
Smalley S., Chalmers A.J, & Morley S.J. (2014). mTOR inhibition and levels of the DNA repair protein MGMT in T98G glioblastoma cells. Molecular Cancer, 13, 144.
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7

Western Blot Analysis of mTOR Pathway

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Cell extracts were prepared by scraping the cells with 60–80 μL of cold lysis buffer (Lee et al., 2014) and storing on ice for 10 min. Clearing of the homogenates, protein quantification, SDS/PAGE, western blotting, and quantification of blots were performed as described previously (Lee et al., 2009). The antibodies (Catalog number) against 4E‐BP1 (#9644), phospho‐4E‐BP1(T37/46) (#2855), phospho‐4E‐BP1(S65) (#9451), phospho‐4E‐BP1(T70) (#9455), 4E‐BP2 (#2845), AKT (#4691), phospho‐AKT(T308) (#2965), phospho‐AKT(S473) (#9271), eIF4A (#2013), eIF4A1 (#2490), eIF4B (#3592), phospho‐eIF4B(S422) (#3591), eIF4E (#2067), eIF4G (#2469), eIF4H (#3469), mLST8 (#3274), Raptor (#2280), Rictor (#2214), p70S6K (#2708), phospho‐p70S6K (T389) (#9205), STAT1 (#9172), phospho‐STAT1(Y701) (#9171), STAT3 (#4904), phospho‐STAT3(Y705) (#9131), phospho‐STAT3(S727) (#9134), mTOR (#2983), phospho‐mTOR(S2448) (#5536), and phospho‐mTOR(S2481) (#2974) were obtained from Cell Signal Technology (Danvers, MA, USA); GAPDH (#CSB‐MA000071M0m) was obtained from Cusabio (Houston, TX, USA).
Lee H., Chin H., Kim H., Jung H, & Lee D. (2020). STAT3‐mediated MLST8 gene expression regulates cap‐dependent translation in cancer cells. Molecular Oncology, 14(8), 1850-1867.
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8

EGCG Mechanism in Cell Signaling

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EGCG (purity>95%) was purchased from Sigma aldrich. Dulbecco's Modified Eagle Medium (DMEM) and foetal bovine serum (FBS) were purchased from Gibco (Logan, UT, USA) and Sijiqing Biological Engineering Materials Co Ltd. (Hangzhou, China), respectively. Bovine serum albumin (BSA) was purchased from Sangon (Shanghai, China). Antibodies against AMPK, phospho-AMPK (T172) and phospho-p70S6K (T389) were purchased from Cell Signaling (Cell Signaling Technology); anti-β-actin antibody was purchased from Santa Cruz Biotech (Santa Cruz Biotechnology); anti-phospho-eEF2 and total-eEF2 was purchased from bioworlde (Guangzhou, China). EGCG was dissolved in distilled water at 100µM, and stored at -20℃ until dilution before use.
Cai Y., Zhao L., Qin Y, & Wu X.Q. (2015). EGCG Blocked Phenylephrin-Induced Hypertrophy in H9C2 Cardiomyocytes, by Activating AMPK-Dependent Pathway. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 19(3), 203-210.
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9

Immunoblotting Assay for Liver Signaling

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Liver tissue was homogenized in RIPA buffer (10mM TRIS pH 8.0, 0.5mM EGTA, 1% Triton X-100, 0.2% SDS, 100mM NaCl) with protease inhibitors cocktail (Roche) and phosphatase inhibitors (2mM sodium orthovanadate, 1mM sodium pyrophosphate, 10mM sodium fluoride, 250nM microcystin). Antibodies used for immunoblotting were: phospho-p70S6K (T389) (Cell Signaling 9206S), phospho-AMPK (T172) (Cell Signaling 2535P), AMPK (Cell Signaling 2793S), p53 (Santa Cruz 6243), acetyl-p53 (K379) (Cell Signaling 2570S), SIRT1 (Millipore 07-131), p21 (Abcam 7960), cleaved CASP3 (Cell Signaling 9664), Cyclin D1 (Santa Cruz 753), and 14-3-3 (Santa Cruz 1657).
Healy M.E., Chow J.D., Byrne F.L., Breen D.S., Leitinger N., Li C., Lackner C., Caldwell S.H, & Hoehn K.L. (2014). Dietary effects on liver tumor burden in mice treated with the hepatocellular carcinogen diethylnitrosamine. Journal of hepatology, 62(3), 599-606.
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10

Western Blot Analysis of Autophagy Markers

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Liver tissues were lysed with a homogenizer in RIPA buffer with protease inhibitors. Total proteins were extracted using centrifugation and denatured via boiling. Then, 50 μg of protein were loaded onto a 12% separation gel and a 5% concentration gel. The total proteins were separated into many bands by electrophoresis and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA) by electroblotting. The membranes were incubated with the rabbit primary antibodies against ASPP2, LC3B (Sigma, MO, USA), p62, Atg7, Beclin-1, CyclinA2, CyclinB1, CyclinE1, phospho-4EBP1 T37/46, phospho-S6 S235/236, and phospho-P70S6K T389 (Cell Signaling, CA, USA). Then, the membranes were incubated with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (Cell Signaling, CA, USA). Finally, immunoreactive bands were developed using a chemiluminescent substrate (Thermo Fisher Scientific, IL, USA). The grayscale was analyzed by ImageJ software, and the relative grayscale value was normalized to that of β-actin.
Shi H., Zhang Y., Ji J., Xu P., Shi H., Yue X., Ren F., Chen Y., Duan Z, & Chen D. (2018). Deficiency of apoptosis-stimulating protein two of p53 promotes liver regeneration in mice by activating mammalian target of rapamycin. Scientific Reports, 8, 17927.
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