Cells were grown in complete medium overnight and treated with DMSO or drugs at recorded concentrations and times. Cells were lysed in RIPA lysis buffer containing Halt protease and phosphatase inhibitors (Thermo Scientific Inc.). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and incubated with total and phosphorylated protein-specific antibodies. Antibodies against p-RB (catalog no. 8180; 1:800 dilution), p-AKT (S473) (catalog no. 9271; 1;1000 dilution), AKT (catalog no. 9272; 1;1000 dilution), p-S6K1 (catalog no. 9234; 1;1000 dilution), S6K1 (catalog no. 2708; 1;1000 dilution), p-S6 (catalog no. 2211; 1;3000 dilution), S6 (catalog no. 2217; 1;3000 dilution), and β-actin (catalog no. 4970; 1;3000 dilution) were obtained from Cell Signaling Technology (Danvers, MA). Binding of the primary antibody was detected using a horseradish peroxidase (HRP)-conjugated secondary antibody and chemiluminescent substrates (Thermo Scientific Inc.). Signal was detected by exposing standard X-ray film. Films were scanned using Epson Perfection V750 pro Scanner. The density of each protein band was quantified by Image J software (NIH). The density of phosphoprotein was normalized to the corresponding total proteins.
P s6k1
Manufactured by Cell Signaling Technology
Sourced in United States
P-S6K1 is a phospho-specific antibody that detects phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr389. S6K1 is a serine/threonine protein kinase that plays a key role in the regulation of cell growth and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
P mtor, by Cell Signaling Technology (14 mentions)
P akt, by Cell Signaling Technology (13 mentions)
P 4ebp1, by Cell Signaling Technology (13 mentions)
4e bp1, by Cell Signaling Technology (7 mentions)
β actin, by Cell Signaling Technology (3 mentions)
P erk1 2, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
P erk, by Cell Signaling Technology (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
P pras40, by Cell Signaling Technology (2 mentions)
Rapamycin, by Merck Group (2 mentions)
P foxo1, by Cell Signaling Technology (2 mentions)
Foxo1, by Cell Signaling Technology (2 mentions)
Jnk1 2, by Cell Signaling Technology (2 mentions)
Sphk1, by Cell Signaling Technology (2 mentions)
Cleaved poly adp ribose polymerase parp, by Cell Signaling Technology (2 mentions)
β tubulin, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
P ampkα, by Cell Signaling Technology (2 mentions)
33 protocols using p s6k1
1
Western Blot Analysis of Protein Phosphorylation
2
Modulation of Cellular Signaling Pathways
3
Immunohistochemical Analysis of Tumor Markers
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Tumor tissue derived from surgical specimens was fixed in 7.5% formalin, and IHC was performed on 4-mm paraffin sections from one representative tissue block per patient following the manufacturer’s instructions. The sections were stained with a monoclonal antibody (mAb) against EGFR, phosphorylated- (p) mTOR, p-4EBPl, and p-S6K1 (1:100 dilution, Cell Signaling [Boston, Massachusetts, USA]) and counterstained with hematoxylin. Protein expression was detected using horseradish peroxidase- (HRP) conjugated rabbit anti-mouse immunoglobulin G (IgG) secondary antibody, followed by colorimetric detection using Diaminobenzidine (DAB). The positive expression was determined under a microscope (×400) by the presence of clear brown granules in the membrane or cytoplasm and/or nucleus. Five fields were selected randomly, and a total of 200 cells were counted. The intensity of staining was scored “−” for colorless, “+” for light yellow, and “++” for yellow and brown–yellow. The positive index scored negative (−) when the number of positive cells was less than 10%, weakly positive (+) when the number of positive cells was 10%–50%, and strongly positive (++) when the number of positive cells was more than 50%.
4
Isolation and Analysis of Cellular Fractions
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Nuclear and cytoplasmic extracts from naive T cells cultured for 3 days were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce), as previously described21 (link). The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against Lamin B1 (D4Q4Z; 1,000×; Cell Signaling Technology). Whole-cell extracts were immunoprecipitated with an anti-Raptor (24C12; 100×; Cell Signaling Technology), anti-Def6 (Rabbit polyclonal; 100×21 (link)), anti-p62 (H-290; 50×; Santa Cruz), anti-TRAF6 (H274; 50×; Santa Cruz), or anti-HA (3F10; 50×; Roche Applied Science) antibodies. Antibodies to p-STAT3 (Y705; 1,000×), p-4E-BP (T37/46; 1,000×), 4E-BP (1,000×), p-S6K1 (S371; 1,000×), S6K1 (1,000×), p-AKT (S473; 1,000×), AKT (1,000×), p-AKT (T308; 1,000×), p-PRAS40 (T246; 1,000×), PRAS-40 (1,000×), p-AMPK (T172; 1,000×), AMPK (1,000×), p-Raptor (S792; 1,000×) and p62 (5114; 1,000×) were obtained from Cell Signaling Technology. Antibodies to IRF4 (M-17; 1,000×), TRAF6 (H274; 500×), and c-Myc (9E10; 500×) were obtained from Santa Cruz. Anti-Bcl6 antibody was obtained from BD (K112-91; 1,000×). Anti-Flag monoclonal antibody M2 (horseradish peroxidase (HRP)) was obtained from Sigma (1,000×).
5
Podocyte Autophagy Regulation Mechanisms
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Aldo, rapamycin (RP), chloroquine (CQ), 3-methyladenine (3-MA), tunicamycin (Tun), tauroursodeoxycholic acid (TAUDC), and anti-β-actin antibody were purchased from Sigma (St Louis, MO). Antibodies against LC3, Akt, p-Akt, mTOR, p- mTOR, S6K1, p-S6K1, 4EBP1, p-4EBP1, GRP78, GRP94, CHOP, FOXO1, p-FOXO1, Rab5, and Rab7 were purchased from Cell Signaling Technology (Beverly, MA). Anti-Podocin, anti-Nephrin, and anti-p62 antibodies were obtained from Abcam (Cambridge, MA). P300, Ac-FOXO1, and nestin antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
6
Immunohistochemical Analysis of Signaling Pathways
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Resected tissue samples were fixed with 10% formalin, routinely embedded in paraffin, cut into 4 μm thick serial sections, and used for H&E and immunohistochemical staining. Immunohistochemical staining was performed using a Roche Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. Primary antibodies against p-AKT (#4060, 1:100; Cell Signaling Technology, Danvers, MA, USA), p-mTOR (clone 49F9, 1:100; Cell Signaling Technology), p-S6K1 (#9204, 1:100; Cell Signaling Technology), p-4EBP1 (clone 236B4, 1:500; Cell Signaling Technology), S100 (polyclonal, Ventana Medical Systems), CD31 (clone JC70A, 1:200, Dako), CD34 (clone QBEnd10, 1:200, Dako), D2-40 (760-4395, Ventana Medical System), and PROX1 (ab199359, 1:500; Abcam, Cambridge, UK) were used. Samples were considered positive when at least 10% of the endothelial cells of abnormal vessels exhibited a signal for the targeted protein.
7
Signaling Pathway Regulation in Cancer
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GDC-0349 was from Dr. Zhou at Hubei Cancer Hospital11 (link). Antibodies of phosphorylated (“p”)-Akt (Ser-473) (#9271), Akt (Thr-308) (#13038), Akt1 (#75692), p-S6K1 (#9234), S6K1 (9202), p-JNK1/2 (#9255), JNK1/2 (#9252), SphK1 (#12071), cleaved-caspase-3 (#9664), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), and β-tubulin (#15115) were purchased from Cell Signaling Tech (Beverly, MA). All cell culture reagents were obtained from Hyclone Co. (Suzhou, China). N-acetylcysteine (NAC), sphingosine-1-phosphate (S1P) and SP600125, rapamycin, perifosine, AZD-2014, puromycin, and polybrene were purchased from Sigma-Aldrich (St. Louis, Mo). Primers, sequences and all viral constructs were designed and provided by Shanghai Genechem (Shanghai, China) unless otherwise mentioned.
8
Cellular Signaling Pathway Analysis in Frozen Muscle
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Frozen gastrocnemius muscle tissue was ground into small pieces in liquid nitrogen and lysed in a lysis buffer containing cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ (Roche Diagnostics, Indianapolis, IN, USA), and then centrifuged. A Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to measure the protein concentration and same concentration of protein of each group was subjected to SDS-PAGE. After transferring, membranes were blocked by 5% skim milk and incubated with a primary antibody overnight. The primary antibodies used for Western blot were p-Akt, Akt, p-mTORc1, mTORc1, p-S6K1, S6K1, p-4E-BP1, 4E-BP1, p-FoxO3a, and FoxO3a (Cell signaling, MA, USA), MuRF1 and Atrogin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (GeneTex, Irvine, CA, USA). The next day, the membranes were incubated with the corresponding secondary antibodies for visualizing the protein bands using LAS3000 luminescent image analyzer (Fuji Film, Tokyo, Japan). β-actin was used as a loading control and Image J software (National Institute of Health, Bethesda, MD, USA) was used for quantification.
9
Western Blot Analysis of Cellular Signaling Pathways
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FLSs were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with phosphatase and protease inhibitors (Roche). Protein extracts were separated by electrophoresis, followed by electrotransfer onto nitrocellulose membrane. After blocking, membranes were incubated with primary antibodies and then exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratory). Specific bands were detected with the enhanced chemiluminescence (ECL) detection kit (Pierce) on Amersham Hyperfilm ECL (GE Healthcare). Reblots were performed using ReBlot Plus Strong Antibody Solution (Millipore). Primary antibodies used were against actin (Cytoskeleton); AKT, p-AKT (S473), p-AKT (T308), p-IKKα/β (S176/180), p-IκB-α (S32/36), IRF1, mTOR, p-mTOR (S2448), p-NF-κB P65 (S536), p-NF-κB P105 (S933), NF-κB P105, p-S6K1 (T389), PTGS2, STAT1, p-STAT1 (Y701), and p-TSC2 (S939) (Cell Signaling Technology); IκB-α, NF-κB P65, and S6K1 (Santa Cruz); SLC38A9 (Sigma); and tubulin (Abcam). FLS lysis and western blotting procedures for SLC38A9 are described elsewhere (Rebsamen et al., 2015 (link)).
10
Immunoblot Analysis of Signaling Pathways
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Immunoblot was performed using a standard protocol as described previously.15 (link),16 (link) Briefly, cells were harvested and protein concentration determined by Bradford assay. Proteins were separated by SDS-PAGE on 12% acrylamide gels and blotted to nitrocellulose membranes. Membranes were incubated with antibodies to DDIT4 (Proteintech #10638-1-AP), hypoxia-inducible factor-1α (HIF-1α) (BD #610959), P-S6RP (Ser240/244; Cell Signalling #2215), P-S6K1 (Thr389; Cell Signalling #9205), S6K1 (Cell Signalling #9202), P-4E-BP1 (Thr37/46, Cell Signalling #9459), 4E-BP1 (Cell Signalling #9452) or actin (Santa Cruz Biotechnology #sc-1616). The secondary anti-rabbit and anti-goat antibodies were obtained from Santa Cruz Biotechnology. Chemiluminescence was used for detection.16 (link)
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