Taselisib, also called GDC-0032, was generated at Genentech, Inc. (South San Francisco, CA). Letrozole was obtained from US Biological. Antibodies used include phospho-AKTSer473, AKT, phospho-PRAS40Thr246, phospho-S6Ser235/236, phospho-S6Ser240/242, S6, phospho-ERKThr202/Tyr204, ERK, phospho-ERαSer118, phospho-ERαSer167, cleaved PARP, p110α, phospho-p70S6KThr389, PR, cyclin E, phospho-mTORSer2448, IGF1R, BRCA1, c-Myc, CAV1, HER2 and cyclin D1 obtained from Cell Signaling (Danvers, MA). Antibodies for ERα and ERβ were obtained from Santa Cruz biotechnology (Santa Cruz, CA) and a βActin antibody was obtained from Sigma (St. Louis, MO).
Phospho p70s6k thr389
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-p70S6K (Thr389) is a lab equipment product that detects the phosphorylation of p70S6 kinase at threonine 389. This phosphorylation site is crucial for the activation of p70S6 kinase, which plays a key role in the regulation of protein synthesis and cell growth.
Automatically generated - may contain errors
Lab products found in correlation
P70s6k, by Cell Signaling Technology (11 mentions)
Phospho mtor ser2448, by Cell Signaling Technology (8 mentions)
Phospho akt ser473, by Cell Signaling Technology (8 mentions)
Total p70s6k, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
P70s6k, by Santa Cruz Biotechnology (3 mentions)
Phospho 4ebp1, by Cell Signaling Technology (3 mentions)
Phospho ampk thr172, by Cell Signaling Technology (3 mentions)
Phospho 4e bp1 thr37 46, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
β actin antibody, by Merck Group (2 mentions)
Phospho mtor, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Total gsk3, by Cell Signaling Technology (2 mentions)
Phospho akt2, by Cell Signaling Technology (2 mentions)
Phospho gsk3 ser9, by Cell Signaling Technology (2 mentions)
Phospho foxo1 3 thr24 32, by Cell Signaling Technology (2 mentions)
Phospho glycogen synthase ser641, by Cell Signaling Technology (2 mentions)
31 protocols using phospho p70s6k thr389
1
Molecular Response to Taselisib and Letrozole
2
Liver Protein Extraction and Western Blot
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Total proteins were extracted from liver tissues using Cell Signaling Lysis Buffer (1M of Tris base pH 7.5, 5M of NaCl, 0.5M of EGTA, 0.5M of EDTA, Triton‐X, 0.1M of Na4P2O7, 1M of β‐glycerophosphate, 1M of Na3VO4) with protease and phosphatase inhibitor cocktails. Protein concentration was quantified using Bradford method as per the manufacturer's instructions. The protein lysates were run on 4%‐12% Bis‐Tris NUPAGE gels (Thermo Fisher Scientific) and transferred on PVDF membrane (Thermo Fisher Scientific). Membranes were blocked with 5% nonfat dry milk for 1 hour prior to overnight incubation at 4°C with primary antibodies as indicated below. Next day, membranes were washed with 1X TBST and incubated with the corresponding secondary antibody. The signal was detected using ECL Substrate solution. GAPDH was used for normalization. Image quantification was performed using Image Studio Lite software (LI‐COR Biosciences). Antibodies used include: Cell Signaling Technology: Phospho‐p70 S6K (Thr389) (Cat# 9205), Phospho‐S6 Ribosomal Protein (Ser235/236) (Cat# 4858S), p70 S6K (Cat# 9202), Anti‐rabbit IgG, HRP‐linked (Cat# 7074), Anti‐mouse IgG, HRP‐linked (Cat# 7076), and GAPDH (D16H11) XP® Rabbit monoclonal (Cat #5174); Santa Cruz Biotechnology: Ribosomal Protein S6 Antibody (C‐8) (Cat# sc‐74459).
3
Myocardial Infarction Protein Analysis
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Myocardial tissue samples were obtained from the infarct’s border zone. Total protein extraction was performed, and protein quantitation was was carried out utilizing a Bicinchoninic acid (BCA) protein assay kit. Equal amounts of protein (30 μg) were resolved by 8% SDS-PAGE and underwent transfer onto polyvinylidene difluoride (PVDF) membranes30 (link). After incubation with rabbit-derived primary antibodies31 (link) targeting caspase (19677–1-AP; Proteintech, China), cleaved caspase3 (9661; Cell Signaling Technology, MA), Bax (50599-2-Ig; Proteintech), Bcl-2 (26593-1-AP; Proteintech), LC3 (14600-1-AP; Proteintech), mTOR (2972; Cell Signaling Technology), phospho-mTOR (5536; Cell Signaling Technology), p70S6k (AF6226; Affinity Biosciences, USA), phospho-p70S6k (Ser371) (9208; Cell Signaling Technology), phospho-p70S6k (Thr389) (9234; Cell Signaling Technology), 4E-BP1 (AF6431; Affinity Biosciences), and phospho-4E-BP1 (2855; Cell Signaling Technology), respectively, the membranes underwent further incubation with secondary antibodies (2 h at ambient). The chemiluminescence HRP substrate (Millipore Corporation, USA) was employed for visualization. A multifunctional imaging analysis system (VersaDoc MP 5000; Bio-Rad, USA) was used for quantitation.
4
Western Blot Analysis of Liver Proteins
5
Mesoglycan Modulates AMPK and mTOR Signaling
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Mesoglycan was kindly provided from Chodang Pham. CO.. Dulbecco’s modified eagle medium (DMEM) and fetal bovin serum (FBS) were purchased from Thermo Scientific (Logan, UT, U.S.A.). Pro-prep protein extract buffer was purchased from Intron Biotechnology (Sungnam, Korea). Antibodies against LKB1, phospho-LKB1 (Ser428), AMPK, phospho-AMPK (Thr172), phospho-ACC (Ser79), mTOR, phospho-mTOR (Ser2448), Bcl-2, Bax, phospho-p70S6K (Thr389) and phospho-4EBP1 (Ser65) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). A monoclonal antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO). Compound C, AMPK inhibitor, was provided by Calbiochem (La Jolla, CA, U.S.A.). Human Platelet-Derived Growth Factor BB was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). AMPK siRNA, Raptor siRNA, Control siRNA, p53, p27, p21, PCNA and Cytochrome C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
6
Phospho-ACC and AMPK Pathway Analysis
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The compounds used in this study were dissolved directly in DMEM and the pH corrected to pH7.4. The phospho-acetyl-CoA carboxylase (ACC) Ser 79 antibody was from the Division of Signal Transduction Therapy at the University of Dundee. The total ACC, total AMPKα, phospho-AMPKα Thr 172, total S6, phospho-S6 Ser 240/244, phospho-p70S6K Thr 389, total IκB, pNF-κB, total IKKα, and total IKKβ antibodies for immunoblotting were from Cell Signaling Technology. Actin antibody was from Merck. Antibodies used in the AMPK activity assays were a generous gift from Prof D. Grahame Hardie at the University of Dundee. Chemical structures were drawn using ChemSketch. BI605906 was a generous gift from Prof Sir Philip Cohen (MRC Protein Phosphorylation and Ubiquitylation Unit, Dundee).
7
Protein Quantification and Western Blot
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Tissue and cell extracts were normalized by measuring total protein concentration using Bio-Rad Dc protein Assay kit according manufacturer's protocol. Extracts were separated by SDS-PAGE, transferred on Immobilon-P membrane (Millipore) and incubated with phosphospecific and protein specific primary and secondary antibodies (Cell Signaling and Santa Cruz Biotechnoly). Due to technical difficulties, instead of stripping and re-probing the same membrane with different antibodies, several gels loaded with the same extracts were run in parallel; after staining with Ponceau S Red and visual examination of the transfer quality, one membrane was stained with phosphor-specific antibodies and another with antibodies specific for the total protein. Experiments with the same extracts were repeated independently at least two times. Following antibodies were used: Cell Signaling Technology: Phospho-p70 S6K (Thr389) #9205, Phospho-p70 S6K (T421/S424) #9204, p70 S6K #9202, Phospho-4E-BP1 (T37/46) #2855, 4E-BP1 #9452, Phospho-S6 (Ser235/236) 32211, Phospho-S6 (Ser240/244) #2215, Anti-Mouse IgG-HRP #7076, aAnti-rabbit IgG-HRP #7074. Santa Cruz Biotechnology: Robosomal Protein S6 sc-74459, Donkey anti-rabbit IgG-HRP sc-2313. Abcam: Anti-S6K1 (phosphor T389) ab2571.
8
Comprehensive Antibody Characterization Protocol
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Antibody for total RB was purchased from BD Biosciences (San Jose, CA, clone C245, catalog #554136). Antibodies purchased from Cell Signaling Technology (Beverly, MA) included: total RB (clone 4H1, Catalog #9309), phospho-RB (Ser608) (#2181), phospho-AKT (Ser473) (#4060), cyclin D1 (#9309), PARP-1 (#9542), caspase 3 (#9665), caspase 9 (#9502), and phospho-p70 S6K (Thr389, #9205). β-actin antibody was purchased from Sigma Aldrich (St. Louis, MO, catalog #A1978).
Laboratory-grade palbociclib was generously provided by Pfizer, Inc. (New York, NY) and purchased from Selleck Chemicals (Boston, MA). Everolimus and pemetrexed were purchased from LC chemical (Woburn, MA). Decitabine was purchased from Sigma (St. Louis, MO). Selumetinib, flavopiridol, sunitinib, AZD5438, ponatinib, AZD4547, erlotinib, pazopanib, imatinib, PF-04691502, dacomitinib and rapamycin were purchased from Selleck Chemicals (Boston, MA). Depsipeptide was obtained from the NCI Repository in the Developmental Therapeutics Program (NSC #309132, Bethesda, MD). Stock solutions for all the agents were prepared in 100% DMSO (Sigma, St. Louis, MO) at 10mM concentrations and stored at -20°C. Stock solutions were diluted in fresh RPMI 1640 media prior to each experiment.
Laboratory-grade palbociclib was generously provided by Pfizer, Inc. (New York, NY) and purchased from Selleck Chemicals (Boston, MA). Everolimus and pemetrexed were purchased from LC chemical (Woburn, MA). Decitabine was purchased from Sigma (St. Louis, MO). Selumetinib, flavopiridol, sunitinib, AZD5438, ponatinib, AZD4547, erlotinib, pazopanib, imatinib, PF-04691502, dacomitinib and rapamycin were purchased from Selleck Chemicals (Boston, MA). Depsipeptide was obtained from the NCI Repository in the Developmental Therapeutics Program (NSC #309132, Bethesda, MD). Stock solutions for all the agents were prepared in 100% DMSO (Sigma, St. Louis, MO) at 10mM concentrations and stored at -20°C. Stock solutions were diluted in fresh RPMI 1640 media prior to each experiment.
9
Western Blot Analysis of Epidermal Proteins
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Mouse tissue (frozen whole skin and muscle samples) and 3D-keratinocytes were homogenized and sonicated in CelLytic MT Mammalian Tissue Lysis/Extraction Reagent (Sigma), and then centrifuged at 16,000g for 10 min at 4°C. The supernatants were harvested, and equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies to KRT5 (Abcam, Cambridge, UK), KRT10 (Abcam), KRT14 (Santa Cruz Biotechnology, Dallas, TX), desmoglein 1 (DSG1; GeneTex, San Antonio, TX), DSG2 (Abcam), desmocollin 3 (DSC3; Santa Cruz Biotechnology), α-tubulin (Cell Signaling), Akt (Cell Signaling), phospho-Akt (Ser473) (Cell Signaling), p70S6K (Cell Signaling), and phospho-p70S6K (Thr389) (Cell Signaling). After washing the membranes with 0.1% Tween 20 in PBS, they were exposed to a horseradish peroxidase-linked anti-rabbit IgG antibody (Cell Signaling). Bands were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) and their intensities were quantified using the program Multi Gauge (Fujifilm, Tokyo, Japan).
10
Protein Signaling Pathway Analysis
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Tissue and cell extracts were prepared in RIPA buffer with Protease/Phosphotase Inhibitor Cocktail, Cell signaling Technology (#5872) according to the manufacturer's protocol. Tissue and cell extracts were normalized by measuring total protein concentration using Bio-Rad Dc protein Assay kit according manufacturer's protocol. Extracts were separated by SDS-PAGE, transferred on Immobilon-P membrane (Millipore) and incubated with phosphospecific and protein specific primary and secondary antibodies. Following antibodies were used: Cell Signaling Technology: Phospho-p70 S6K (Thr389) #9205, Phospho-p70 S6K (T421/S424) #9204, p70 S6K #9202, Phospho-S6 (Ser235/236) 32211, Phospho-S6 (Ser240/244) #2215, Anti-Mouse IgG-HRP #7076, aAnti-rabbit IgG-HRP #7074. Santa Cruz Biotechnology: Robosomal Protein S6 sc-74459, Donkey anti-rabbit IgG-HRP sc-2313. Abcam: Anti-S6K1 (phosphor T389) ab2571.
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