A total 60 µg of proteins were separated on 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis, (PA003D; Auragene, Changsha, People’s Republic of China) transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) and probed with primary antibodies: anti-LC3B antibody (Abcam, Hong Kong), anti-Beclin1 antibody (Epitomics, Hong Kong), anti-STMN1 antibody (Abcam), anti-phosphoinositide 3-kinase (anti-PI3K) antibody (Santa Cruz Biotechnology, Dallas, TX, USA), anti-P-PI3K (Santa Cruz Biotechnology), anti-mammalian target of rapamycin (mTOR) antibody (Abcam), anti-p-mTOR (Abcam), anti-S6K (Abcam), anti-P-S6K antibody (Abcam), or anti-β-actin antibody (Boster, Wuhan, People’s Republic of China) at 4°C for one night, and followed by secondary antibodies conjugated with horseradish peroxidase at room temperature for 1 hour. The protein bands were visualized by the Amersham ECL system (RPN998, GE, Fairfield, CN, USA) and scanned. Data was analyzed by densitometry using Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) normalized to β-actin expression.
Anti s6k
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-S6K is a primary antibody that binds to S6K, a serine/threonine protein kinase involved in the regulation of cell growth and proliferation. This antibody can be used for the detection and study of S6K in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti β actin antibody, by Boster Bio (1 mentions)
Anti lc3b antibody, by Abcam (1 mentions)
Anti stmn1 antibody, by Abcam (1 mentions)
Anti p pi3k, by Santa Cruz Biotechnology (1 mentions)
Amersham ecl system, by GE Healthcare (1 mentions)
Anti p mtor, by Abcam (1 mentions)
Image pro plus software 6, by Media Cybernetics (1 mentions)
Endothelial cell growth supplement (ecgs), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Anti phospho akt s473, by Cell Signaling Technology (1 mentions)
Fitc phalloidin, by Merck Group (1 mentions)
Anti fak, by Cell Signaling Technology (1 mentions)
Anti akt1, by Proteintech (1 mentions)
Ku 0063794, by Merck Group (1 mentions)
Anti phospho fak y397, by Cell Signaling Technology (1 mentions)
Vegf a, by Thermo Fisher Scientific (1 mentions)
Anti phospho s6k t389, by Cell Signaling Technology (1 mentions)
Anti raptor, by Cell Signaling Technology (1 mentions)
Anti rictor, by Cell Signaling Technology (1 mentions)
5 protocols using anti s6k
1
Western Blot Analysis of Autophagy Proteins
2
mTOR Modulation Impacts Endothelial Angiogenesis
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Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-AktS473, anti-phospho-S6KT389, anti-FAK, anti-phospho-FAKY397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).
3
Drosophila Ovary Protein Quantification
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Drosophila ovaries were homogenized in RIPA buffer containing Complete Protease Inhibitors and Phosphatase Inhibitors (Roche). Antibodies were used at the following concentrations: rabbit anti-P-S6K T398 (1:1,000) (Cell Signaling), guinea pig anti-S6K (1:10,000) (PMID: 20444422), guinea pig anti-Nprl3 (1:1,000) (37 (link)), mouse anti-actin (1:10,000)(Abcam), rabbit anti-GFP11 (1:1,000) (Bonopus), rat anti-OLLAS (1:1,000) (Novus), mouse anti-T7 (1:1,000) (Millipore sigma), and rabbit anti-GFP (1:1,000) (Cell signaling). The band intensity was quantified using Image J analysis tool (NIH).
4
Signaling Pathways Analysis in Cell Culture
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All chemicals were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. CYT was purchased from the ShanghaiPureone Biotechnology Co., Ltd (Shanghai, China). Anti-5-HT1A and anti-BDNF antibodies were purchased from Chemicon (Temecula, CA, USA). Anti-mTOR, p-mTOR, anti-AKT, p-AKT, anti-S6K, p-S6K, anti-CREB, and p-CREB antibodies were purchased from Abcam (Cambridge, UK). Anti-β-actin antibody was purchased from Sigma. All of the chemicals and reagents used were commercially available and of standard biochemical quality.
5
Western Blot Analysis of Cancer Cell Signaling
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BGC-823, MGC-803, MKN-45 cells and mouse tumor tissues were lysed with RIPA buffer (Solarbio, Shanghai, China) supplemented with Protease Inhibitor Cocktail and Protein Phosphatase Inhibitor (Solarbio, Shanghai, China). The concentration of protein samples was determined by using the BCA Protein Quantitation Kit (Promega, USA). Samples were separated by SDS-PAGE; proteins were then transferred onto Nitrocellulose membrane filters (Millipore, USA) by wet blotting. After treatment with skim milk, membranes were incubated with primary antibody overnight at 4°C and then with corresponding secondary antibody for 1 h at room temperature. The Odyssey system (LI-COR, USA) was used to measure the protein signal.
Anti-KLF2 (1:1000, ab194468), anti-Caspase3 (1:1000, ab44976), anti-CCND1 (1:1000, ab134175), anti-p27 (1:1000, ab32034), anti-p16 (1:1000, ab201980), anti-active Caspase-3 (1:1000, ab2302), anti-Cleaved PARP (1:1000, ab32064), anti-PARP (1:1000, ab32138), anti-S6K (1:1000, ab32529), and anti-p-S6K (1:1000, ab126818) were purchased from Abcam (Cambridge, MA, USA), and anti-GAPDH (1:3000, #5174), anti-p-AKT (1:1000, #9271), anti-AKT (1:1000, #9272), anti-PTEN (1:1000, #9552), anti-mTOR (1:1000, (#2972) and anti-p-mTOR (1:1000, #5536) were purchased from Cell Signaling Technology (Danvers, MA, USA).
Anti-KLF2 (1:1000, ab194468), anti-Caspase3 (1:1000, ab44976), anti-CCND1 (1:1000, ab134175), anti-p27 (1:1000, ab32034), anti-p16 (1:1000, ab201980), anti-active Caspase-3 (1:1000, ab2302), anti-Cleaved PARP (1:1000, ab32064), anti-PARP (1:1000, ab32138), anti-S6K (1:1000, ab32529), and anti-p-S6K (1:1000, ab126818) were purchased from Abcam (Cambridge, MA, USA), and anti-GAPDH (1:3000, #5174), anti-p-AKT (1:1000, #9271), anti-AKT (1:1000, #9272), anti-PTEN (1:1000, #9552), anti-mTOR (1:1000, (#2972) and anti-p-mTOR (1:1000, #5536) were purchased from Cell Signaling Technology (Danvers, MA, USA).
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