Thapsigargin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin was from Invitrogen (Carlsbad, CA, USA). DMEM and FBS were purchased from GIBCO Invitrogen (Carlsbad, CA, USA). The Hoechst kit and Lyso-Tracker Red probe for acidic lysosome staining were from Beyotime (Haimen, Jiangsu, China). Anti-PERK, Anti-BiP, anti-p-eIF2α, anti-eIF2α, anti-cleaved-caspase 3, anti-LC3, anti-p-AKT, anti-p-p70S6K1, anti-p70S6K1, anti-p-4EBP1 and anti-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ATF-6, anti-p62 and anti-GAPDH were from Abcam (Cambridge, UK). The CHOP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were obtained from Sigma-Aldrich with the highest purity available.
Anti p70s6k1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p70S6K1 is a primary antibody that recognizes the p70 ribosomal protein S6 kinase 1 (p70S6K1). p70S6K1 is a serine/threonine kinase that plays a role in the regulation of cell growth, proliferation, and protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Anti mtor, by Cell Signaling Technology (7 mentions)
Anti p p70s6k1, by Cell Signaling Technology (6 mentions)
Anti 4e bp1, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Anti phospho p70s6k1, by Cell Signaling Technology (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Anti p 4ebp1, by Cell Signaling Technology (3 mentions)
Anti phospho mtor, by Cell Signaling Technology (3 mentions)
Anti p mtor, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti ampk, by Cell Signaling Technology (2 mentions)
Anti raptor, by Cell Signaling Technology (2 mentions)
Anti rictor, by Cell Signaling Technology (2 mentions)
Anti p ampk, by Cell Signaling Technology (2 mentions)
Anti acc, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Anti s6, by Cell Signaling Technology (2 mentions)
16 protocols using anti p70s6k1
1
Thapsigargin and Rapamycin Induced Cell Stress Response
2
Quantitative Immunoblot Analysis of Signaling Proteins
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Protein extracts from tumor tissues and cells were prepared in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Sigma-Aldrich). Protein samples were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Primary antibodies (1:500 dilution) used here are as follows: anti-Akt, anti-Akt (ser473), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-phospho-p70S6K1 (Thr389), anti-p70S6K1, anti-phospho-4EBP (Thr37/46), anti-4EBP (Cell Signaling Technology, Beverly, MA, USA), anti-α-synuclein (Abcam, Cambridge, MA, USA), and anti-β-actin (Sigma-Aldrich). Blots were developed with an enhanced chemiluminescence system (GE Healthcare Biosciences, Piscataway, NJ, USA). Densitometric analysis of the blots were performed using the Quantity One software (Bio-Rad Laboratories, Richmond, CA, USA).
3
Western Blot Analysis of mTORC1 Signaling
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Cells were washed with PBS and harvested in RIPA buffer. Lysates were centrifuged at 14,000 g for 10 min at 4 °C and protein extracts were measured by Bradford assay. Next, 30 μg of protein was subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 3% not-fat dry milk in Tris-buffered saline containing 0.01% Tween 20 (TBST) buffer and then incubated with the indicated antibodies overnight at 4 °C.
The antibodies for the mTORC1 signaling pathway were purchased from Cell Signaling Technology as follows: anti-phospho AMPK (#2535), anti- AMPK (#5831), anti-phospho Thr389 p70-S6K1 (#9234), anti- p70-S6K1 (#9202), anti-phospho Thr37/46 4EBP1 (#2855), anti- 4EBP1 (#9644), anti-phospho mTOR (#2971), and anti-mTOR (#2983). Anti-HIV-1 p24 antibody (ab9071) was purchased from Abcam. Additionally, the anti-GAPDH antibody (sc-47724) used as an internal control was purchased from Santa Cruz Biotechnology Inc. Specific bands were detected using HRP-labeled anti-mouse or anti-rabbit IgG, and the reactions were developed using the enhanced chemiluminescence system (Bio-Rad). The protein band density was quantified using ImageJ and normalized to the GAPDH control.
The antibodies for the mTORC1 signaling pathway were purchased from Cell Signaling Technology as follows: anti-phospho AMPK (#2535), anti- AMPK (#5831), anti-phospho Thr389 p70-S6K1 (#9234), anti- p70-S6K1 (#9202), anti-phospho Thr37/46 4EBP1 (#2855), anti- 4EBP1 (#9644), anti-phospho mTOR (#2971), and anti-mTOR (#2983). Anti-HIV-1 p24 antibody (ab9071) was purchased from Abcam. Additionally, the anti-GAPDH antibody (sc-47724) used as an internal control was purchased from Santa Cruz Biotechnology Inc. Specific bands were detected using HRP-labeled anti-mouse or anti-rabbit IgG, and the reactions were developed using the enhanced chemiluminescence system (Bio-Rad). The protein band density was quantified using ImageJ and normalized to the GAPDH control.
4
Investigating PI3K/Akt/mTOR Signaling Cascade
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Cells were lysed using complete radioimmune precipitation assay (RIPA) buffer supplemented with complete ULTRA protease inhibitor cocktail tablets (Roche, Basel, Switzerland) and sodium orthovanadate. Anti-Akt2, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Pras40, anti-phospho-Pras40 (Thr246), anti-Gsk3A, anti-phospho-Gsk3 (Ser21/9), anti-Tsc2, anti-phospho-Tsc2 (Thr1426), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-p70S6k1, anti-phospho-p70S6k1 (Thr389), anti-p70S6K2, anti-4E-BP1, anti-4E-BP1 (Thr37/46) (Cell Signalling Technology, Danver, Massachusetts, USA) rabbit polyclonal antibodies were used in immunoblotting. Luciferase constructs (pLightSwitch_3′UTR) (Switchgear genomics, Carlsbad, CA, USA) containing the 3′ UTR region of Akt2, Pras40, Gsk3a, CSF1 and Itgb1 was individually transfected into HEK293T cells using pGL4.12 (luc2CP) as a normaliser. Luciferase activity was measured by using the dual luciferase assay (Promega).
5
Osteoclastogenesis and Bone Resorption Analysis
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Glutathione S-transferase (GST)-receptor activator of nuclear factor-κB ligand (RANKL) vector and PLAT-E cells were obtained from Dr. S.L. Teitelbaum (Washington University, St. Louis, MO, USA) and T. Kitamura (Tokyo University, Tokyo, Japan), respectively. pMSCVpuro-Cre (#34564) was obtained from Addgene (Watertown, MA, USA). Recombinant mouse RANKL and macrophage colony-stimulating factor (M-CSF) were purchased from R&D Systems (Minneapolis, MN, USA). C-terminal Peptide of Type I Collagen (CTx) Enzyme-linked immunosorbent assay (ELISA) kit was obtained from Immunodiagnostic Systems (Boldon, UK). Antibodies were from the following companies: anti-β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-mTOR, anti-Raptor, anti-Rictor, anti-Hamartin, anti-p-p70S6K1, and anti-p70S6K1 were from Cell Signaling Technology (Danvers, MA, USA). THUNDERBIRD SYBR quantitative polymerase chain reaction (qPCR) Mix was supplied by TOYOBO (Osaka, Japan). Other chemicals used were all of the highest purity commercially available.
6
Anticancer compound analysis protocol
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Anti-PARP, anti-caspase 3, anti-ACC, anti-p-AMPK, anti-AMPK, anti-p-mTOR, anti-mTOR, anti-p-P70S6K1, anti-P70S6K1, anti-cyclin D1, anti-cyclin E1, anti-p53 and ant-β actin antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-CDK2, anti-CDK6, anti-Bcl2, anti-BAX, and p21 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgGs were purchased from Zymed Laboratories (San Francisco, CA, USA). The compound C was purchased from Calbiochem (San Diego, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), propidium iodide (PI), JC-1 and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HsA was kindly provided by Prof. Jong Rok, Lee (Daegu Haany University, Gyeongsan, Korea), and isolated as previously described [23 (link)]. The pure HsA (1.31 g) was isolated from the chloroform extract (150 g) of H. lyrata, and was stored at −20 °C. The purity (>97%) was verified by comparing retention time with standard compounds.
7
Protein Quantification and Phosphorylation Assay
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Cultured cells were solubilized in lysis buffer containing 1% Nonidet P‐40 detergent. Samples were then subjected to SDS/PAGE, followed by transfer to polyvinylidene fluoride membranes and subsequent immunoblotting with anti‐β‐Actin (#sc‐47778) (Santa Cruz), anti‐p‐p70S6K1 (#9234), and anti‐p70S6K1 (#2708) (Cell Signaling Technology, Danvers, MA, USA).
8
Reagents and Antibodies for Cell Signaling Analysis
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Dimethyl sulfoxide (DMSO), bicinchoninic acid (BCA), trichloroacetic acid (TCA), sulforhodamine B (SRB), and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), trypsin-EDTA solution (1X), antibiotic-antimycotic solution (100X) and phosphate-buffered saline (PBS) (1X) were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-p-AMPKα, anti-AMPKα, anti-p-ACC, anti-ACC, anti-p-mTOR, anti-mTOR, anti-p-4EBP1, anti-4EBP1, anti-p-eIF4E, anti-eIF4E, anti-p-P70S6K1, anti-P70S6K1, anti-p-RPS6, anti-RPS6, anti-rictor, anti-p-Akt, anti-Akt, and anti-E-cadherin were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-PKC-α, anti-p-Rac1, anti-Rac1, anti-PCNA, anti-β-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology. (Santa Cruz, CA, USA). Anti-p-rictor was purchased from Millipore (Temecula, CA, USA). Anti-p-PKC-α, anti-F-actin, and anti-Ki-67 were purchased from Abcam (Cambridge, MA, USA). Gefitinib was purchased from Selleckchem (Houston, TX, USA) and rapamycin was purchased from Tocris Bioscience (Bristol, UK).
9
Cellular Responses to Pharmacological Agents
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Fetal bovine serum (FBS), cell culture media, penicillin/streptomycin, and all other reagents used for cell culture studies were purchased from Welgene (Gyeongsan, Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI), 5-Fu, rhodamine 123 (Rh123), CoCl2, compound C, crystal violet solution, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Akt, anti-p-Akt, anti-p-AMPK, anti-Bcl-2, anti-caspase-3, anti-cleaved caspase-3, anti-p-ERK, anti-PARP, anti-mTOR, anti-p-mTOR, anti-p70S6K1, anti-p-p70S6K1, and anti-p-4E-BP1 antibodies were supplied by Cell Signaling Technologies (Danvers, MA, USA). Anti-β-actin and anti-vascular endothelial growth factor (VEGF) antibodies, horseradish peroxidase (HRP)-conjugated goat anti-rabbit, and goat anti-mouse IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-hypoxia-inducible factor-1α (HIF-1α) antibody was obtained from BD Biosciences (SanJose, CA, USA). Dimethyl sulfoxide (DMSO) was purchased from AppliChem (Darmstadt, Germany) and Junsei Chemical Co. (Tokyo, Japan).
10
Intestinal Protein Extraction and Immunoblotting
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Total proteins from intestinal epithelial cells or intestinal organoids were extracted with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) with proteinase inhibitor (Roche) and phosphatase inhibitor (Thermo Fisher Scientific) and then subjected to SDS-polyacrylamide gel electrophoresis, followed by transferring to polyvinylidene difluoride membranes and subsequent immunoblotting assay. Quantification was performed with Li-COR analysis system (LI-COR Bioscience). The antibodies used were anti-phospho-p70S6K1 (Cell Signaling Technology), anti-p70S6K1 (Cell Signaling Technology), anti-phospho-S6 (Cell Signaling Technology), anti-S6 (Cell Signaling Technology), anti-β-actin (Abcam).
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