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P s6kt389

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P-S6KT389 is an antibody that detects phosphorylation of the S6 kinase (S6K) at threonine 389. S6K is a key regulator of cell growth and proliferation, and its phosphorylation at Thr389 is a marker of mTOR pathway activation.

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Lab products found in correlation

Pakt s473, by Cell Signaling Technology (14 mentions) Ps6 s240 244, by Cell Signaling Technology (5 mentions) Pakt t308, by Cell Signaling Technology (5 mentions) 4e bp1, by Cell Signaling Technology (5 mentions) Ps6 s235 236, by Cell Signaling Technology (4 mentions) P 4ebp1 s65, by Cell Signaling Technology (4 mentions) Total s6k, by Cell Signaling Technology (4 mentions) Total akt, by Cell Signaling Technology (4 mentions) Perk t202 y204, by Cell Signaling Technology (3 mentions) P 4e bp1 t37 46, by Cell Signaling Technology (3 mentions) Gapdh, by Cell Signaling Technology (3 mentions) β actin, by Santa Cruz Biotechnology (3 mentions) Actin, by Merck Group (3 mentions) Actin, by Cell Signaling Technology (3 mentions) Pampkt172, by Cell Signaling Technology (2 mentions) Paccs79, by Cell Signaling Technology (2 mentions) P mtors2448, by Cell Signaling Technology (2 mentions) Cd41 fitc, by BioLegend (2 mentions) Annexin 5 fitc, by BioLegend (2 mentions) Rat insulin elisa kit, by ALPCO (2 mentions)

21 protocols using p s6kt389

1

Analyzing AMPK and mTOR Signaling in CD4+ T Cells

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CD4+ T cells were isolated from lymph nodes with CD4+ T cells isolation kit (Stem cell). After pretreatment with Compound C or AICAR for 30 minutes, CD4+ T cells were stimulated with PMA/Ionomycin for another 20 minutes. Cells were lysed in buffers with protease and phosphorylation inhibitors. Protein concentrations were determined by BCA assay (Thermo Scientific). The antibodies of p-AMPKT172 (#4188), p-ACCS79 (#3661), p-S6S235/236 (#4803), p-S6S240/244 (#4803), p-ERKT202/Y204 (#4370), p-4EBP1T37/46 (#7547), p-AKTS473 (#4060), p-S6KT389 (#9205) were purchased from Cell Signaling Technology. β-actin was used as a loading control.
Rao E., Zhang Y., Li Q., Hao J., Egilmez N.K., Suttles J, & Li B. (2016). AMPK-dependent and independent effects of AICAR and compound C on T-cell responses. Oncotarget, 7(23), 33783-33795.
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2

Comprehensive Antibody Reagents for Cellular Signaling Analysis

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Antibodies against pS6K (T389) (#9205), S6K (#9202), pS6 (S240/244) (#2215), S6 (#2217), pAkt (T308) (#13038), pAkt (S473) (#4060), AKT (4691), p4E-BP1 (S65) (#9456), 4E-BP1 (#9644), RagA (#4357), RagB (#8150), RagC (#9480), Sestrin2 (#8487), MIOS (#13557), EEA1 (#3288, used for immunofluorescence (IF)), Lamp1 (#9091, used for IF) and Rab7 (#9367, used for IF) were purchased from Cell Signaling Technology. Lamp2 mouse monoclonal antibody (H4B4, for human cells) and Lamp2 rat-monoclonal antibody (GL2A7, for mouse cells) for IF were obtained from Abcam. Anti-FLAG (F1804, used for IF), anti-HA (3F10), and anti-PMP70 (SAB4200181, used for IF) were obtained from Sigma. WDR24 (20778-1-AP, used for IP) and Sestrin2 (10795-1-AP, used for IP) antibody was purchased from Proteintech. Antibodies against SZT2 (sc-242153), NPRL2 (sc-376986), SEC13 (sc-514308) and WDR59 (sc-137927, only used in immunoblotting of WDR59 in MEFs) were obtained from Santa Cruz. SEH1L (ab187307) antibody was form Abcam. The rabbit WDR59 monoclonal antibody used in IF experiments (1:100, validated in this study with knockout cells, Extended Data Fig. 5b) and immunoblotting experiments (1:1000) in HEK293T was kindly provided by Jianxin Xie from Cell Signaling Technology. This antibody only recognized WDR59 from human cells, but not MEFs. Anti-NPRL3 (NBP1-88447, used for IP) was obtained from Novus Biologicals.
Peng M., Yin N, & Li M.O. (2017). SZT2 Dictates GATOR Control of mTORC1 Signaling. Nature, 543(7645), 433-437.
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3

Comprehensive Protein Expression Analysis

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Commercial antibodies were used to detect PPP6C (rabbit, A300-844A; Bethyl Laboratories, Inc.), α-tubulin (mouse, DM1A; Sigma-Aldrich), MVH (rabbit, ab13840; Abcam), γH2AX (rabbit, 9718; Cell Signaling Technology, Inc.), p-CHK1 (S345) (rabbit, BS4041; Bioworld Technology, Inc.), p-CHK2 (T68) (rabbit, BS4043; Bioworld Technology, Inc.), p-p53 (S15) (rabbit, 12571; Cell Signaling Technology, Inc.), CHK1 (rabbit, BS1052; Bioworld Technology, Inc.), p-AKT (S473) (rabbit, 4060; Cell Signaling Technology, Inc.), p-AMPK (T172) (rabbit, 2535; Cell Signaling Technology, Inc.), p-mTOR (S2448) (rabbit, 5536; Cell Signaling Technology, Inc.), p-S6K (T389) (rabbit, 9234; Cell Signaling Technology, Inc.), p-rpS6 (S240/244) (Rabbit, 5364; Cell Signaling Technology, Inc.), GAPDH (rabbit, 5174; Cell Signaling Technology, Inc.) and β-actin (mouse, sc-47778, Santa Cruz). Secondary antibodies were purchased from ZhongShan Golden Bridge Biotechnology Co., LTD (Beijing).
Hu M.W., Meng T.G., Jiang Z.Z., Dong M.Z., Schatten H., Xu X., Wang Z.B, & Sun Q.Y. (2016). Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity. PLoS Genetics, 12(12), e1006513.
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4

Western Blotting Analysis of Intracellular Signaling

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Cultured cells were washed in cold PBS and lysed in buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate and 25% glycerol. Protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to 0.2 μm nitrocellulose membrane. After blocking with 0.1% casein, membranes were probed with antibodies to p4E-BP1 T36/47 (#9459), Akt (#9272), pAkt T308 (#13038), pAkt S473 (#9271), pmTOR S2448 (#2983), PARP (#9542), S6 (#2217), pS6 S235/236 (#4858), pS6 S240/244 (#2215), pS6K T389 (#9234) (Cell Signaling Technologies, Boston, MA, USA) and actin (#A5441) (Sigma-Aldrich Co., LLC, St Louis, MO, USA). The signals were visualized by Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA).
Woo S.U., Sangai T., Akcakanat A., Chen H., Wei C, & Meric-Bernstam F. (2017). Vertical inhibition of the PI3K/Akt/mTOR pathway is synergistic in breast cancer. Oncogenesis, 6(10), e385-.
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5

Western Blot Analysis of Cell Signaling

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Cells were lysed and proteins were extracted in RIPA buffer that was supplemented with phosphatase and protease inhibitors. Proteins were separated by SDS–polyacrylamide gel electrophoresis gels and transferred to a PVDF (polyvinylidene difluoride) membrane. Membranes with blocked with 5% bovine serum albumin and probed using specific antibodies. Actin (1:2,000), pAKT (S473) (1:1,000), pS6K (T389) (1:1,000), HA (1:1,000) were all from Cell Signaling Technology (CST).
Osmanbeyoglu H.U., Toska E., Chan C., Baselga J, & Leslie C.S. (2017). Pancancer modelling predicts the context-specific impact of somatic mutations on transcriptional programs. Nature Communications, 8, 14249.
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6

Characterization of TSC2 Signaling Pathway

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P-TSC2 S939 (LS-C358381) was from Biozol (Sontheim, Germany). Antibodies specific for TSC2/tuberin (#4308), P-AKT S473 (#4060), AKT (#4691), P-S6K T389 (#9234), P-S S235/6 (#2211), S6 (#2217), P-ERK T202/Y204 (#4370), ERK (#4696), P-4E-BP T37/46 (#2855), as well as MG132 and cyclohexamide were from Cell Signaling (Danvers, MA, USA). Involucrin (SY8; ab20202) was from Abcam (Cambridge, UK), Actin antibody (A1978) was from Sigma-Aldrich (St. Louis, MO, USA) and LAMP-2 antibody (sc-18822) from SantaCruz Biotechnology (Dallas, TX, USA). LY294002, U0126 were from were from Calbiochem (San Diego, CA, USA).
Ferreri A., Lang V., Kaufmann R, & Buerger C. (2022). mTORC1 Activity in Psoriatic Lesions Is Mediated by Aberrant Regulation through the Tuberous Sclerosis Complex. Cells, 11(18), 2847.
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7

Quantitative Platelet and Pancreatic Assays

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Purified GP2B3A complex (MBS135714), mouse rCela2a protein (MBS1246487), human rCELA2A (MBS1090462) and the CELA2A ELISA kit (MBS932150) were purchased from MyBiosource. CELA2A antibody (SAB1104798) and the elastase substrate N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide substrate (S8511) were purchased from Sigma-Aldrich. The FITC-conjugated PAC1 antibody (340507, BD Biosciences) and tetramethylrhodamine ethyl ester (TMRE) reagent (T669, Thermo Fisher Scientific), Annexin V-FITC (640905, Biolegend), CD41-FITC (303703, Biolegend) were used for platelet aggregation analysis. Total AKT (CST, 9272), pAKT (S473) (CST, 9271s), pAKT (T308) (CST, 9275s), total S6k (CST, 9202), pS6k (T389) (CST, 9206), total IRS (CST, 2382), pIRS (Y608), (CST, 2385S), pIRS (S636/S639) (CST, 2388s), Gapdh (CST, 3683s), and Actin (CST, 4970) antibodies were purchased from Cell Signaling Technologies. Recombinant A1AT (alpha1 antitrypsin, ab91136), A1AT antibody (ab9400) and ITGA2B antibodies (ab63983) were purchased from Abcam. Human c-peptide ELISA kit (80-CPTHU-E01.1, ALPCO), Human Glucagon ELISA kit (10-1271-01, Mercodia), Rat c-peptide ELISA (80-CPTRT-E01, ALPCO), Mouse Ultrasensitive Insulin ELISA (80-INSMSU-E01, ALPCO), Human Insulin ELISA kit (21-IAAHU-E01, ALPCO), Rat Insulin ELISA kit (80-INSRT-E01, ALPCO) was applied for C-peptide, glucagon, and insulin measurements.
Esteghamat F., Broughton J.S., Smith E., Cardone R., Tyagi T., Guerra M., Szabó A., Ugwu N., Mani M.V., Azari B., Kayingo G., Chung S., Fathzadeh M., Weiss E., Bender J., Mane S., Lifton R.P., Adeniran A., Nathanson M.H., Gorelick F.S., Hwa J., Sahin-Tóth M., Belfort-DeAguiar R., Kibbey R.G, & Mani A. (2019). CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nature genetics, 51(8), 1233-1243.
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8

Comprehensive Antibody Panel for IMPDH1/2

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Anti-IMPDH1 (WH0003614M1), BrdU (BMC9318) antibodies (Sigma-Aldrich); anti-IMPDH2 (ab131158), β-actin (ab6276), p21 (ab109199), GAPDH (ab9485), HPRT1 (ab10479) antibodies (Abcam); anti-β-actin (#3700), p-Akt (T308, #4056), (S473, #4060), p-S6K (T389) (#9234), p-ERK (#4370), p-AMPK (#2535), p-p53 (#9284), Nucleostemin (#9495) antibodies (Cell Signaling Technology); anti-pan-Akt (sc-81434), p53 (sc-126), Nucleolin (sc-13057) antibodies (Santa Cruz Biotechnology); anti-GMPS antibody (A302–417A)(Bethyl laboratory); anti-HPRT1 (VMA00483) antibody (Bio-Rad); anti-Nucleostemin (MAB4311) antibody (Millipore); anti-Vinculin (BZL03106) antibody (Biozol Diagnostica).
Kofuji S., Hirayama A., Eberhardt A.O., Kawaguchi R., Sugiura Y., Sampetrean O., Ikeda Y., Warren M., Sakamoto N., Kitahara S., Yoshino H., Yamashita D., Sumita K., Wolfe K., Lange L., Ikeda S., Shimada H., Minami N., Malhotra A., Morioka S., Ban Y., Asano M., Flanary V.L., Ramkissoon A., Chow L.M., Kiyokawa J., Mashimo T., Lucey G., Mareninov S., Ozawa T., Onishi N., Okumura K., Terakawa J., Daikoku T., Wise-Draper T., Majd N., Kofuji K., Sasaki M., Mori M., Kanemura Y., Smith E.P., Anastasiou D., Wakimoto H., Holland E.C., Yong W.H., Horbinski C., Nakano I., DeBerardinis R.J., Bachoo R.M., Mischel P.S., Yasui W., Suematsu M., Saya H., Soga T., Grummt I., Bierhoff H, & Sasaki A.T. (2019). IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma. Nature cell biology, 21(8), 1003-1014.
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9

Antibodies for Cell Signaling Analysis

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p-H2A.X, H2A.X, p-CAD (S1859), CAD, p-ERK (T202/Y204), ERK1/2, p-S6K (T389) and S6K antibodies were purchased from Cell Signaling Technology. P-CAD (T456) and DHODH antibodies were obtained from Santa-Cruz Biotechnology. An Alexa-Fluor 647-conjugated p-H2A.X (Ser139) antibody was purchased from BD Biosciences.
Brown K.K., Spinelli J.B., Asara J, & Toker A. (2017). Adaptive Reprogramming of De Novo Pyrimidine Synthesis is a Metabolic Vulnerability in Triple-Negative Breast Cancer. Cancer discovery, 7(4), 391-399.
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10

Quantitative Platelet and Pancreatic Assays

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Purified GP2B3A complex (MBS135714), mouse rCela2a protein (MBS1246487), human rCELA2A (MBS1090462) and the CELA2A ELISA kit (MBS932150) were purchased from MyBiosource. CELA2A antibody (SAB1104798) and the elastase substrate N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide substrate (S8511) were purchased from Sigma-Aldrich. The FITC-conjugated PAC1 antibody (340507, BD Biosciences) and tetramethylrhodamine ethyl ester (TMRE) reagent (T669, Thermo Fisher Scientific), Annexin V-FITC (640905, Biolegend), CD41-FITC (303703, Biolegend) were used for platelet aggregation analysis. Total AKT (CST, 9272), pAKT (S473) (CST, 9271s), pAKT (T308) (CST, 9275s), total S6k (CST, 9202), pS6k (T389) (CST, 9206), total IRS (CST, 2382), pIRS (Y608), (CST, 2385S), pIRS (S636/S639) (CST, 2388s), Gapdh (CST, 3683s), and Actin (CST, 4970) antibodies were purchased from Cell Signaling Technologies. Recombinant A1AT (alpha1 antitrypsin, ab91136), A1AT antibody (ab9400) and ITGA2B antibodies (ab63983) were purchased from Abcam. Human c-peptide ELISA kit (80-CPTHU-E01.1, ALPCO), Human Glucagon ELISA kit (10-1271-01, Mercodia), Rat c-peptide ELISA (80-CPTRT-E01, ALPCO), Mouse Ultrasensitive Insulin ELISA (80-INSMSU-E01, ALPCO), Human Insulin ELISA kit (21-IAAHU-E01, ALPCO), Rat Insulin ELISA kit (80-INSRT-E01, ALPCO) was applied for C-peptide, glucagon, and insulin measurements.
Esteghamat F., Broughton J.S., Smith E., Cardone R., Tyagi T., Guerra M., Szabó A., Ugwu N., Mani M.V., Azari B., Kayingo G., Chung S., Fathzadeh M., Weiss E., Bender J., Mane S., Lifton R.P., Adeniran A., Nathanson M.H., Gorelick F.S., Hwa J., Sahin-Tóth M., Belfort-DeAguiar R., Kibbey R.G, & Mani A. (2019). CELA2A mutations predispose to early-onset atherosclerosis and metabolic syndrome and affect plasma insulin and platelet activation. Nature genetics, 51(8), 1233-1243.
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