Specific antibodies for pAkt(Ser473)(#4060, 1:5000), pAkt(Thr308)(#4056, 1:2500), Akt(#4691, 1:5000), pINSR(#3024, 1:5000), INSRβ(#3025, 1:5000), p-p70-S6K(#97596, 1:2000), S6K(#9202, 1:5000), pFoxO1(S256)(#9461, 1:2000), FoxO1(#2880, 1:5000), pGSK3β(#5558, 1:5000), GSK3α/β(#5676, 1:5000), p-ERK1/2(T202/Y204)(#9101, 1:5000), ERK1/2(#4695, 1:5000), and Hsp70(#4872, 1:2000) were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). pIRS1(#09432, 1:2500) and IRS1(#06248, 1:2500) were acquired from (Merck KGaA, Darmstadt, Germany). β-Actin(#A228, 1:5000) was procured from (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies Anti-Rabbit IgG (#NA934, 1:5000) and Anti-Mouse IgG (#NA931, 1:5000) were purchased from (GE Healthcare BioSciences AB, Chicago, IL, USA). Anti-Rabbit IgG Secondary Antibody (Alexa Fluor™ 568, 1:5000) was obtained from (Thermo Fisher Scientific, Waltham, MA, USA).
P p70s6k
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, China
P-p70S6K is a lab equipment product that detects the phosphorylated form of the p70S6 kinase protein. It serves as a tool for researchers to measure the activation state of this important signaling molecule.
Automatically generated - may contain errors
Lab products found in correlation
P70s6k, by Cell Signaling Technology (126 mentions)
P mtor, by Cell Signaling Technology (82 mentions)
P akt, by Cell Signaling Technology (78 mentions)
P 4ebp1, by Cell Signaling Technology (63 mentions)
4e bp1, by Cell Signaling Technology (39 mentions)
P ampk, by Cell Signaling Technology (24 mentions)
Bcl 2, by Cell Signaling Technology (23 mentions)
Gapdh, by Cell Signaling Technology (22 mentions)
Cleaved caspase 3, by Cell Signaling Technology (20 mentions)
β actin, by Cell Signaling Technology (19 mentions)
Pvdf membrane, by Merck Group (16 mentions)
P erk, by Cell Signaling Technology (15 mentions)
β actin, by Santa Cruz Biotechnology (13 mentions)
Beclin 1, by Cell Signaling Technology (13 mentions)
β actin, by Merck Group (13 mentions)
P erk1 2, by Cell Signaling Technology (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Caspase 3, by Cell Signaling Technology (10 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (10 mentions)
Erk1 2, by Cell Signaling Technology (9 mentions)
207 protocols using p p70s6k
1
Immunoblotting for Insulin Signaling Pathway
2
Galangin-Modulated Apoptotic Signaling
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Galangin was purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). The primary antibodies against Bcl-2, p-Akt, t-Akt, p-p70S6K, t-p70S6K, Bax, p21, p53, DR5, caspase-3, -7, -8 and -9 were purchased from Cell Signaling Technology, Inc. (Dancers, MA, USA). The primary antibodies against Gapdh, cmyc and p53 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
3
Western Blot Analysis of Cell Signaling
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Western blot protein assays were conducted as previously reported [20 ]. Cells were treated with omipalisib and harvested by trypsinization, followed by resuspension in lysis buffer (containing 1 mM PMSF and 0.5 mM PhosSTOP) with brief sonication. The protein concentrations were determined with a protein assay kit according to the manufacturer’s instructions (Beyotime, P0010, China). Cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes (GE Healthcare). After blocking with blocking buffer containing 5% nonfat milk, membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary antibody. Blots were imaged with an imaging system (MYECL, Thermo scientific). Primary antibodies, AKT (#9272), ERK (#4695), P70S6K (#9202), S6 (#2217), 4EBP1 (#9644), p-AKT (Ser473, #4060), p-ERK (Thr202/Tyr204, #4376), p-P70S6K (Ser371, #9208), p-S6 (Ser235/236, #2211), p-4EBP1 (Thr37/46, #2855), and CDKN1B (#3688) were purchased from Cell Signaling Technology. CCND1 (ab134175) was obtained from Abcam.
4
Comprehensive Evaluation of PMF Efficacy
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Chromatographical acetonitrile was bought from Thermo Fisher Scientific (China). Analytical-grade petroleum ether (PE), methanol, and ethyl acetate were bought from Honeywell (USA). Ultrapure water was obtained from a Milli-Q system (Millipore, USA). The other chemicals were analysis-grade chemicals.
RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Logan, UT, USA). Cell counting kit 8 (CCK-8) was bought from DOJINDO (Japan). Antibodies (including β-actin, p-AMPK, AMPK, p-S6, S6, p-P70S6K, P70S6K, p-p53, p53, and COX-2) were purchased from Cell Signaling Technology (USA); Compound C (an AMPK inhibitor) was bought from Santa Cruz Biotech (USA). Matrix basement membrane was purchased from Corning Co., Ltd. (USA). The apoptosis-Hoechst Staining Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Crystal violet reagent was purchased from Damao (Tianjin, China). Ki-67 was purchased from Wuhan Servicebio Technology Co., Ltd. The four PMFs obtained from CRCP were used in DMSO and were stored at −20°C and diluted for use.
RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Logan, UT, USA). Cell counting kit 8 (CCK-8) was bought from DOJINDO (Japan). Antibodies (including β-actin, p-AMPK, AMPK, p-S6, S6, p-P70S6K, P70S6K, p-p53, p53, and COX-2) were purchased from Cell Signaling Technology (USA); Compound C (an AMPK inhibitor) was bought from Santa Cruz Biotech (USA). Matrix basement membrane was purchased from Corning Co., Ltd. (USA). The apoptosis-Hoechst Staining Kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Crystal violet reagent was purchased from Damao (Tianjin, China). Ki-67 was purchased from Wuhan Servicebio Technology Co., Ltd. The four PMFs obtained from CRCP were used in DMSO and were stored at −20°C and diluted for use.
5
Nobiletin Modulates Cellular Metabolism
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Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Yeasen Biotechnology Co., Ltd. Cell counting kit 8 (CCK8) was purchased from DOJINDO (Japan). Antibodies against cleaved‐caspase 3, p‐AMPK, p‐S6, p‐P70S6K, and SIRT1 were purchased from Cell Signaling Technology (USA); anti‐AMPK and β‐actin were purchased from Santa Cruz Biotech (USA). Antibody against PARP‐2 was purchased from Abcam (UK). Nobiletin was purchased from Chengdu Must Bio‐Technology Co., Ltd (Sichuan, China) and dissolved in dimethyl sulfoxide and stored at −20°C until diluted upon use. Recombinant adenovirus vectors expressing green fluorescent protein (Ad‐Flag) and Flag‐tagged PARP‐2 (Ad‐PARP‐2) were purchased from Genechem Co., Ltd. (Shanghai, China). The viruses were extended in HEK293A cells and purified by virus purification kit (Biomiga, USA), then dialyzed in dilution buffer and stored at −80°C.
6
Molecular Signaling Pathways in Neurological Research
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Primary antibodies were purchased as follows: P-PRAS40 (Thr246; cat. no. 2997; Cell Signaling Technology, Inc.), PRAS40 (cat. no. 2691; Cell Signaling Technology, Inc.), p-mTOR (Ser2448; cat. no. 5536; Cell Signaling Technology, Inc.), mTOR (cat. no. 2983; Cell Signaling Technology, Inc.), p-AKT (Ser473; cat. no. 4060; Cell Signaling Technology, Inc.), AKT (cat. no. 4685; Cell Signaling Technology, Inc.), p-P70S6K (Thr389; cat. no. 9234; Cell Signaling Technology, Inc.), P70S6K (cat. no. 14130; Cell Signaling Technology, Inc.), Light chain 3-I/II (LC3-I/II; cat. no. 12741; Cell Signaling Technology, Inc.), 14-3-3 (cat. no. 8312; Cell Signaling Technology, Inc.), P62 (cat. no. 18420-1-AP; ProteinTech Group, Inc.) and GAPDH (cat. no. sc-365062; 1:2,000; Santa Cruz Biotechnology, Inc.). Goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (cat. no. sc-2054; Santa Cruz Biotechnology, Inc.). The following drugs were obtained from commercial sources as follows: Pilocarpine (cat. no. S4231; Selleck Chemicals), lithium chloride (cat. no. L9650; Sigma-Aldrich; Merck KGaA), Scopolamine (cat. no. S2508; Selleck Chemicals) and LY3023414 (cat. no. S8322; Selleck Chemicals).
7
Western Blot Analysis of Cellular Signaling
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Cell lysates were extracted with CETi protein extraction solution (TransLab, Daejeon, Korea). Thirty to fifty micrograms of protein extract was used for western blotting with antibodies against p-Rbs, p21, cyclin D1, GSK3β, p-GSK3β, CatB, GS, p-GS, p38MAPK, p-p38MAPK, p70S6K, p-p70S6K (Cell Signaling, Denvers, MA, USA), α-tubulin, mouse IgG peroxidase conjugate, rabbit IgG peroxidase conjugate (Sigma-Aldrich Corp.) and Rb (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The human phospho-MAPK Array Kit (R&D Systems, Inc., Minneapolis, MN, USA) was used according to the manufacturer's instructions. A polyclonal antibody against CST1 was obtained from mice immunized with the purified protein.
8
Analysis of p70S6K Activation in B Cells
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CD19+ B cells were isolated from the spleens of B6 mice, treated with CBirTox and then washed, and subsequently cultured with B6.CBir1 Tg CD4+ T cells for 48 hours. CD4+ T cells were then removed from the culture using BD IMag CD4+ magnetic beads, and the B cells were washed 3 times before being lysed. Protein concentrations were determined using the Micro BCA Protein Assay Kit (Pierce). Equal amounts of protein were loaded onto a 10% Tris-HCl gel and separated by SDS-PAGE. Protein was then transferred to immobilon-P transfer membranes (Millipore) and detected with the following antibodies: p70S6K, pp70S6K (Thr421/Ser424) (Thr389), and HRP-linked rabbit or mouse anti-IgG (Cell Signaling). Densitometry was performed using an AlphaImager 2000 (Alpha Innotech).
9
Curcumin Modulates Signaling Pathways in Cells
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For Western blot analysis, cells were seeded in a 6-well plate at a seeding density of 1 × 106 cells/well and treated with curcumin concentrations for 48 h. Cells treated with 0.1% DMSO were used as a negative control. Cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, Gillingham, UK). All samples were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) nitrocellulose membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% BSA in Tris-buffered saline supplemented with 0.1% Tween-20 buffer (Sigma-Aldrich, Gillingham, UK). Western blot analyses were performed using primary antibodies against p-NF-κB (#3033), p-P70S6K (#9204), cleaved caspase-3 (#9661), p-Akt (#4060) and β-actin (#3700) (Cell Signaling Technology, Danvers, MA, USA). The signals were detected using AmershamTM ECLTM Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Amersham, UK). Protein bands were visualized by Molecular Imager ChemiDocTM XRS+ Imaging System (Bio-Rad, Hercules, CA, USA) and analyzed by Image Lab software (version 6.0) (Bio-Rad, Hercules, CA, USA).
10
Western Blot Analysis of Mouse Tissue Lysates
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Protein lysates from mouse tissue samples were generated using M-PER mammalian extraction reagent (Thermo Scientific, USA) and separated by electrophoresis in discontinuous SDS-polyacrylamide gels. The following antibodies were used for immuno detection: pERK1/2 (Cell Signaling, USA), ERK1/2 (Cell Signaling, USA), c-MYC (Cell Signaling, USA), Cleaved-Caspase-3 (Cell Signaling, USA), pP70S6K (Cell Signaling, USA), P70S6K (Cell Signaling, USA), and anti-ACTIN (MP Biomedicals, USA).
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