Phospho s6k1

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-S6K1 is a primary antibody that specifically recognizes the phosphorylated form of ribosomal protein S6 kinase 1 (S6K1). S6K1 is a serine/threonine protein kinase that plays a critical role in the regulation of cell growth, proliferation, and protein synthesis.

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Lab products found in correlation

Close spelling variants of this product

Phospho-S6K1 (Thr389) (12 citations) Anti-phospho-S6K1 (10 citations) Phospho-S6K1 T389 (5 citations) Anti-phospho (T389) p70 S6K1 (3 citations) Anti-phospho-S6K1 (T389) antibody (2 citations) Anti-phospho p70 S6K1 (Thr389) (2 citations) Phospho-S6K1 Thr308 (2 citations)

13 protocols using phospho s6k1

Quantifying mTOR Signaling Pathway

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phospho‐S6K1 (T389, Cat#: 9205), phospho‐S6K1 (T421/S424, Cat#: 9204), phos‐rpS6 (S235/236, Cat#: 4858), phos‐rpS6 (S240/244, Cat#: 5364), rpS6 (Cat#: 2317), 4E‐BP1 (Cat#: 9644), phos‐mTOR (S2448, Cat#: 5536), mTOR (Cat#: 2972), phos‐Akt (S473, Cat#: 4060), phos‐Akt (T308, Cat#: 5106), Akt (pan, Cat#: 4691), phos‐Akt1 (S473, Cat#: 9018), Akt1 (Cat#: 2967) were from Cell Signaling Technology. glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; Cat#: sc‐32233) and S6K1 (Cat#: sc‐230) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IRDye 800CW Goat anti‐Mouse IgG (Cat#: 926‐32210) and IRDye 680LT Goat anti‐Rabbit IgG (Cat#: 926‐68021) were from LI‐COR Biosciences.

Miyazaki M., Moriya N, & Takemasa T. (2020). Transient activation of mTORC1 signaling in skeletal muscle is independent of Akt1 regulation. Physiological Reports, 8(19), e14599.

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Whole Cell Lysate Preparation and Immunoprecipitation

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To prepare whole cell lysates, cells or tissues were lysed in Buffer B as described previously [7 (link)]. For immunoprecipitations, 200 μg whole cell lysate was diluted in IP buffer and immunoprecipitated with 4G10 anti-phosphotyrosine agarose conjugate (05-777, Millipore) as described previously [9 (link)]. Whole cell lysates and immunoprecipitates were processed by WES to separate and visualize cellular proteins according to a standard instrument protocol. Primary antibodies used were: Ron β (#sc-374626, 1:25), GAPDH (#sc-25778, 1:300), PCNA (#sc-56, 1:25) from Santa Cruz Biotechnology; pan AKT (#4691, 1:25), phospho-AKT (#9271, 1:25), S6 (#2217, 1:25), phospho-S6 (#4856, 1:25), phospho-eIF4B (#3591, 1:25), phosphor-AKT1(# 9018, 1:25), phosphor-AKT2 (# 8599, 1:25), AKT1(# 2938, 1:25), AKT2 (# 3063, 1:25), phospho-S6K1 (#9234, 1:25), S6K1 (#2708, 1:25), cleaved PARP (#9541, 1:25), phospho-GSK3α/β (#9331, 1:25), GSK3α/β (#5676, 1:25), Met (#8198, 1:25), Ret (#14556, 1:25) and Axl (#8661, 1:25) from Cell Signaling Technology. Secondary antibodies were included in a Wes Master Kit (PS-MK14, ProteinSimple).

Wang L., Wang L., Cybula M., Drumond-Bock A.L., Moxley K.M, & Bieniasz M. (2020). Multi-kinase targeted therapy as a promising treatment strategy for ovarian tumors expressing sfRon receptor. Genes & Cancer, 11(3-4), 106-121.

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Western Blot Analysis of Protein Signaling

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Protein lysates were prepared in RIPA lysis buffer, and separated on 10% SDS–polyacrylamide gel electrophoresis gels by electrophoresis. Subsequently, the lysates were transferred to nitrocellulose membranes and probed with the following antibodies and concentrations: HIF-2α 1:1,000 (Novus #NB100-122), HIF-1α 1:1,000 (Cayman Chemicals #1006421), caspase-3 1:1,000 (Cell Signaling #9662 Danvers, MA, USA), GAPDH 1:2,000 (Cell Signaling #2118), β-tubulin 1:1,500 (Cell Signaling #2146), phospho-4E-BP1 1:1,000 (S65, Cell Signaling #9451), 4E-BP1 1:1,000 (Cell Signaling #9452), phospho-S6K1 1:1,000 (T389, Cell Signaling #9205), c-Myc 1:5,000 (Abcam #32072), S6K1 1:1,000 (Cell Signaling #2708), phospho-AKT 1:1,000 (S473, Cell Signaling #9271), AKT 1:1,000 (Cell Signaling #9272), phospho-EGFR 1:1,000 (Y1068, Cell Signaling #3777), EGFR 1:1,000 (Cell Signaling #4267) ANO1 1:500 (Abcam ab64085), phospho-CAMKIIα 1:1,000 (T286, Cell Signaling #12716) and CAMKII 1:1,000 (Cell Signaling #11945). Uncropped immunoblot images are included in Supplementary Fig. 8.

Nakazawa M.S., Eisinger-Mathason T.S., Sadri N., Ochocki J.D., Gade T.P., Amin R.K, & Simon M.C. (2016). Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth. Nature Communications, 7, 10539.

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Immunoblotting of mTOR Pathway Proteins

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The phospho and total antibodies used for immunoblotting were purchased from Cell Signaling (Beverly, CA): phospho‐Akt (Ser³⁰⁸; 1:1000), phospho‐mTOR (Ser²⁴⁴⁸; 1:500), phospho‐S6K1 (Thr³⁸⁹;1: 500), phospho‐4E‐BP1 (Thr^37/46; 1:1000), and phospho‐rpS6 (Ser^240/244; 1:250). Total protein was detected for Akt (1:1000), mTOR (1:500), S6K1 (1:500), 4E‐BP1 (1:1000), and rpS6 (1:250). Anti‐rabbit IgG HRP‐conjugated secondary antibody was purchased from Amersham Bioscience (1:2000).

Walker D.K., Drummond M.J., Dickinson J.M., Borack M.S., Jennings K., Volpi E, & Rasmussen B.B. (2014). Insulin increases mRNA abundance of the amino acid transporter SLC7A5/LAT1 via an mTORC1‐dependent mechanism in skeletal muscle cells. Physiological Reports, 2(3), e00238.

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Comprehensive Antibody Panel for Cellular Signaling

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S6K1 antibody, phospho-S6K1 (Thr389) antibody, phospho-S6K1 (Thr421/Ser424) antibody, 4E-BP1 antibody, phospho-4E-BP1(Thr37/46) antibody, phospho-4E-BP1 (Ser65) antibody, phospho-4E-BP1 (Thr70) antibody, eIF4E antibody, phospho-eIF4E (Ser209) antibody, p44/42 MAP kinase antibody, phospho-p44/42 MAP kinase (Thr202/Tyr204) antibody, phospho-Akt (Ser473) antibody, Akt antibody, phospho-PKA substrate (RRXS*/T*) (100G7E) antibody, phospho-(Ser/Thr) PKA substrate antibody, c-Myc antibody, cyclin B1 antibody, survivin antibody, β-actin antibody, and goat-rabbit IgG conjugated with HRP were purchased from Cell Signaling Technology. Cyclin D3 rabbit polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). CD3 antibody (FITC-conjugated), CD13 antibody (PE conjugated), CD19 antibody (perCP conjugated), CD33 antibody (PE conjugated), CD34 antibody (PE-conjugated), CD41a antibody (PE-conjugated), CD42a antibody (FITC conjugated), CD42b antibody (FITC conjugated), CD71 antibody (FITC conjugated) and CD235a (FITC conjugated) were purchased from Becton Dickinson (BD).

Li C.L., Yang J.G., Lin D., Zhao Y.S., Liu S., Xing S.N., Zhao S., Chen C.Q., Jiang Z.M., Pu F.F., Cao J.P, & Ma D.C. (2014). Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines. PLoS ONE, 9(12), e114389.

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Cell Signaling Pathway Analysis Protocol

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Rotenone, CAT, poly-D-lysine (PDL), H₂DCFDA, 2′7′-dichlorofluorescein (DCF), protease inhibitor cocktail, TTFA, antimycin A, and DAPI, and EGTA were purchased from Sigma (St Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), 0.05% Trypsin-EDTA, NEUROBASAL^TM Media, and B27 Supplement were purchased from Invitrogen (Grand Island, NY, USA). Horse serum and fetal bovine serum (FBS) were supplied by Hyclone (Logan, UT, USA). Enhanced chemiluminescence solution was from Millipore (Billerica, MA, USA). CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay kit was from Promega (Madison, WI, USA). BAPTA/AM was purchased from Calbiochem (San Diego, CA, USA). Mito-TEMPO and KN93 were acquired from ALEXIS Biochemicals Corporation (San Diego, CA, USA). The following antibodies were used: phospho-CaMKII (Thr286), phospho-S6K1 (Thr389), phospho-4E-BP1 (Thr70), 4E-BP1, cleaved-caspase-3, PARP (Cell Signaling Technology, Beverly, MA, USA); S6K1, CaMKII (Santa Cruz Biotechnology, Santa Cruz, CA, USA); β-tubulin, phospho-mTOR (Ser2448), mTOR, HA, FLAG (Sigma); Goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, and rabbit anti-goat IgG-HRP (Pierce, Rockford, IL, USA). Other chemicals were purchased from local commercial sources and were of analytical grade.

Liu C., Ye Y., Zhou Q., Zhang R., Zhang H., Liu W., Xu C., Liu L., Huang S, & Chen L. (2016). Crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis. Oncotarget, 7(7), 7534-7549.

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Antibodies and Small Molecules for ER Stress

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The anti-RNF5 antibody was previously described (Bromberg et al., 2007 (link); Delaunay et al., 2008 (link)). Other antibodies were obtained from the following sources: SLC1A5 (Santa Cruz sc-99002 and Sigma HPA035240), vimentin (Sigma HPA001762), SLC38A2 (Santa Cruz sc-67081 and Sigma HPA035180), phospho-mTOR (2971), phospho-S6K1 (9206), phospho-S6 (4857), phospho-4EBP1 (9459), mTOR (2972), S6K1 (9202), 4EBP1 (9644), phospho-eIF2α (9721), eIF2α (9722), IREα (3294), PDI (3501), Ero1-Lα (3264), calnexin (2433) (all from Cell Signaling Technology), BiP (Santa Cruz sc-13968), phospho-IREα (Novusbio NB100-2323), p62 (BD Biosciences 610832), V5 (Invitrogen 46-0705), and Flag (Sigma F1804). Brefeldin A was purchased from eBiosciences. Tunicamycin, thapsigargin, cisplatin, doxorubicin, and paclitaxel were purchased from Sigma.

Jeon Y.J., Khelifa S., Ratnikov B., Scott D.A., Feng Y., Parisi F., Ruller C., Lau E., Kim H., Brill L.M., Jiang T., Rimm D., Cardiff R.D., Mills G.B., Smith J.W., Osterman A.L., Kluger Y, & Ronai Z.A. (2015). Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-inducing Chemotherapies. Cancer cell, 27(3), 354-369.

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Western Blot Analysis of mTOR Pathway Proteins

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Cells or tissues were harvested, lysed in a RIPA buffer with protease inhibitors and phosphatase inhibitors, and then subjected to western blotting as described^{15 (link)}, using various antibodies as follows: RPS27L polyclonal rabbit antibody was raised and purified as described^{13 (link)}, Phospho-4E-BP1, 4E-BP1, Phospho-S6K1 (Thr389), Phospho-AKT (Ser473), AKT, Phospho-S6, S6, β-TrCP, DEPTOR, mTOR, RICTOR, RAPTOR, GβL, PARP, cleaved caspase-3 (Cell Signaling Technology, MA, USA), β-actin, HA, LC3-I/II (Sigma, MO, USA), S6K1, mTOR (Santa Cruz Biotechnology, CA, USA), and p62 (MBL Life science, Japan).

Xiong X., Liu X., Li H., He H., Sun Y, & Zhao Y. (2018). Ribosomal protein S27-like regulates autophagy via the β-TrCP-DEPTOR-mTORC1 axis. Cell Death & Disease, 9(11), 1131.

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Western Blot Antibody Panel for Signaling Pathways

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The following antibodies were purchased from Cell Signaling and used at the indicated dilution for Western blot analysis: S6K1 (#9202, 1 : 1000), phospho-S6K1 (#9234, 1 : 1000), AKT (#9272S, 1 : 1000), phospho-AKT S437 (#4058S, 1 : 1000), 4EBP1 (#9452, 1 : 1500), phospho-4EBP1 (#9451, 1 : 1000) ULK (8054S, 1 : 1000), phospho-ULK S757 (#6888, 1 : 1000), FoxO3a (12829S, 1 : 1000), phospho-FoxO1(T24)/FoxO3a(T32) (9464T, 1 : 1000), GSK3β (12456S, 1 : 1000), phospho-GSK3α/β S21/9 (12456S, 1 : 1000), TSC2 (3635S, 1 : 1000), phospho-TSC2 (3617S, 1 : 1000), mTOR (#2983, 1 : 1000), Raptor (#2280, 1 : 1000), mlST8 (#3274, 1 : 1000), DEPTOR (#11816, 1 : 1000), PRAS40 (#2691, 1 : 1000), Sin1 (#12860S, 1 : 1000), phospho-CREB (#9198S, 1 : 1000), CREB (#9197S, 1 : 1000), phospho-Thr (#9381S, 1 : 1000), phospho-β-catenin (#9561, 1 : 1000), PKA Cat α (#4782, 1 : 1000), phospho-glycogen synthase (#3891, 1 : 1000), and actin (#3700, 1 : 1000). Flag (#F3165, 1 : 10 000) was obtained from Sigma. HA (#sc-7392 or #sc-805, 1 : 500) and Myc (#sc-40, 1 : 1000), FKBP12 (#sc-133067, 1 : 1000) were obtained from Santa Cruz. EGFP (#632380, 1 : 2000) was from Clontech. Horseradish peroxidase (HRP) linked secondary antibodies (#NXA931V anti-mouse, 1 : 8000 or #NA934V anti-rabbit, 1 : 4000) were from GE Healthcare.

Melick C.H, & Jewell J.L. (2020). Small molecule H89 renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors. Biochemical Journal, 477(10), 1847-1863.

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Regulation of H3K9me3 and AP-1 by Akt1

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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Welgene Corporation (Daegu, Republic of Korea). HaCaT cells were grown in DMEM, supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 U/ml). HaCaT cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂. 12-O-tetradecanoylphorbol-13-acetate (TPA) and FLAG-agarose bead were purchased from Sigma (St. Louis, MO, USA). Polybrene, recombinant Akt1 and a polyclonal antibody that detects H3K9me3/p-H3S10 were purchased from Millipore (Billerica, MA, USA). Lentiviral helper plasmids (pMD.2G and psPAX.2) were acquired from Addgene (Cambridge, MA, USA). pGL3-AP-1-luciferase vector was generated by cloning 3x AP-1 consensus sequence DNA and Renilla luciferase reporter plasmid (pGL4.74) was acquired from Promega (Madison, WI, USA). Human H3.3, HP1γ and 14-3-3 cDNAs were amplified from human cDNA library by PCR and subcloned into pcDNA3 plasmid. LY294002, rapamycin, rabbit polyclonal antibodies against c-Jun, c-Fos, HP1γ, Akt1, phospho-Akt1, S6K, phospho-S6K1, HA, DYKDDDDK (FLAG) tag were purchased from Cell Signaling Technology (Beverly, MA, USA). Total actin antibody was acquired from Developmental Studies Hydridoma Bank (Iowa City, IA, USA)

, & Choi W.J. (2014). The Heterochromatin-1 Phosphorylation Contributes to TPA-Induced AP-1 Expression. Biomolecules & Therapeutics, 22(4), 308-313.

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