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Anti phospho rps6s240 244

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Anti-phospho-rpS6S240/244 is a lab equipment product that recognizes the phosphorylation of ribosomal protein S6 at serine residues 240 and 244. This antibody can be used to detect the activation state of the mTOR signaling pathway.

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Lab products found in correlation

Ecl substrate, by GE Healthcare (2 mentions) Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions) Ab109199, by Santa Cruz Biotechnology (1 mentions) Ab108349, by Abcam (1 mentions) β actin, by Merck Group (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Anti fkbp51, by Cell Signaling Technology (1 mentions) Anti ndrg1, by Cell Signaling Technology (1 mentions) Anti p70 s6 kinase, by Cell Signaling Technology (1 mentions) Anti fkbp52, by Cell Signaling Technology (1 mentions) Anti phospho akt s473, by Cell Signaling Technology (1 mentions) Nupage system, by Thermo Fisher Scientific (1 mentions) Anti gapdh am4300, by Thermo Fisher Scientific (1 mentions) Anti rps6, by Cell Signaling Technology (1 mentions) Anti raptor, by Cell Signaling Technology (1 mentions) Horseradish peroxidase coupled anti rabbit or anti mouse secondary antibodies, by Cell Signaling Technology (1 mentions) Image lab software, by Bio-Rad (1 mentions) Ripa buffer, by Merck Group (1 mentions) Chemidoc mp imaging system, by Bio-Rad (1 mentions) Anti p16ink4a, by Abcam (1 mentions)

5 protocols using anti phospho rps6s240 244

1

Western Blot Analysis of Cellular Proteins

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Total protein extracts from cultured cells were obtained using Ripa Buffer (50 mM TrisHCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, and 0.1% SDS) containing protease and phosphatase inhibitor cocktails (Sigma). Equal amounts of proteins (20 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes by western blotting. Membranes were then blocked and incubated with the following primary antibodies: anti-P16 (ab108349, Abcam), anti-P21 (ab109199), anti-SOD2 (sc-30080, Santa Cruz Biotechnology), anti-phospho-rpS6S240/244 (number 5364, Cell Signaling Technology), and B-actin (A2228, Sigma) for loading control. Detection was performed using horseradish peroxidase-coupled anti-mouse or anti-rabbit secondary antibodies (Cell Signaling), ECL substrate (GE Healthcare), and conventional developing using X-ray films. The intensity of the bands was determined by densitometry using Totalab TL120 software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom).
Piccinato C.A., Sertie A.L., Torres N., Ferretti M, & Antonioli E. (2015). High OCT4 and Low p16INK4A Expressions Determine In Vitro Lifespan of Mesenchymal Stem Cells. Stem Cells International, 2015, 369828.
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2

Evaluation of mTOR Pathway Activation

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Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane using the Invitrogen Nu-Page system (Carlsbad, CA). Western blot analysis was performed using the following protein-/phospho-protein-specific antibodies: anti-p70 S6 kinase (#2708), anti-phospho-p70 S6 kinase (T389) (#9234), anti-rpS6 (#2217), anti-phospho-rpS6(S240/244) (#5364), anti-phospho-NDRG-1 (T346) (#5482), anti-NDRG-1 (#9485), anti-phospho-Akt (S473) (#4060), anti-Akt (#4691), anti-FKBP51 (#12210), anti-FKBP52 (#11826), anti-mTOR (#2972), anti-Rictor (#9476), anti-phospho-Rictor (T1135) (#3806), and anti-Raptor (#2280) were from Cell Signaling Technologies (Danvers, MA). Anti-FKBP25 (ab16654) and anti-FKBP12 (ab2918) were from Abcam. Anti-GAPDH (AM4300) was from Ambion Austin, TX.
Schreiber K.H., Ortiz D., Academia E.C., Anies A.C., Liao C.Y, & Kennedy B.K. (2015). Rapamycin-mediated mTORC2 inhibition is determined by the relative expression of FK506-binding proteins. Aging Cell, 14(2), 265-273.
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3

Rapamycin-Mediated Senescence Pathway

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Total protein extracts from rapamycin-treated and untreated (DMSO) cells were obtained at passages when untreated cells entered senescence and stop proliferating and at passages when rapamycin-treated cells entered into proliferative arrest using Ripa Buffer (Sigma, cat. R0278) containing protease and phosphatase inhibitor cocktails (Sigma Aldrich, cat. P8340 and P5726). Equal amounts (20μg) of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were then blocked and incubated with the following primary antibodies: anti-P16INK4A (Abcam, ab108349), anti-phospho-RPS6S240/244 (Cell Signaling Technology, #5364), and ß-actin (Sigma, A2228) for loading control. Detection was performed using horseradish peroxidase-coupled anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology, #7074 and #7076), ECL substrate (GE Healthcare, cat. RPN2236), and the ChemiDoc MP Imaging System (Bio-Rad). The intensity of the bands was determined by densitometry using The Image Lab Software (Bio-Rad). The immunoblotting experiments were repeated more than three times and similar results were observed.
Antonioli E., Torres N., Ferretti M., Piccinato C.D, & Sertie A.L. (2019). Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells. PLoS ONE, 14(1), e0204784.
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4

Comprehensive Analysis of mTOR Pathway

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Following infection and other treatments, total cell lysates were prepared for SDS-PAGE and Western blotting as described [72 (link)]. Primary antibodies anti-mTOR (#2983), anti-phospho-S6K1 (T389; #9234), anti-phospho-RPS6 (S235/236; #2211), anti-phospho-RPS6 (S240/244; #5364), anti-phospho-eIF4B (S422, #3591), anti-eIF4B (#3591), anti-phospho-4E-BP1 (T37/46; #2855), anti-4E-BP1 (#9644), anti-phospho-eIF2α (S51; #3398), anti-eIF2α (#2103), anti-PABCP1 (#4992), and β-actin (#3700) were purchased from Cell Signaling Technologies; anti-phospho-PKR (EIF2AK2) (T451; #07–886) was obtained from Millipore; anti-RPS6 (#sc-74459), anti-S6K1 (#sc-230), and anti-PKR (EIF2AK2) (#sc-6282) were acquired from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-linked goat anti-rabbit and goat anti-mouse IgG secondary antibodies were purchased from Sigma-Aldrich.
Chaparro V., Leroux L.P., Masvidal L., Lorent J., Graber T.E., Zimmermann A., Arango Duque G., Descoteaux A., Alain T., Larsson O, & Jaramillo M. (2020). Translational profiling of macrophages infected with Leishmania donovani identifies mTOR- and eIF4A-sensitive immune-related transcripts. PLoS Pathogens, 16(6), e1008291.
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5

Proteomic Analysis of 293FT Cells

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The 293FT cell line, high glucose DMEM media, penicillin-streptomycin-glutamine mixture (PSG), MEM non-essential amino acids solution (NEAA), sodium pyruvate, G418 and Alexa Fluor® 568 goat anti-mouse IgG (H+L) antibody were from Life Technologies. Normal fetal bovine serum (FBS), protease inhibitor cocktail, phosphatase inhibitor cocktails (2 & 3), HPLC grade acetonitrile (ACN), trifluoroacetic acid (TFA) and formic acid (FA) were from Sigma. The dialyzed FBS, 13C615N4 L-arginine, 4,4,5,5-D4 L-lysine and DMEM media deficient in arginine and lysine were from Thermo Fisher Scientific. Sequence grade trypsin, treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK), was from Promega. Primary antibody to myosin heavy chain (MF20) was from R&D systems. The PPP1R12A antibody (H-130) was from Santa Cruz. The β-actin antibody was from Cell Signaling. The 40S ribosomal protein S6 (RPS6) antibody was from Santa Cruz. The anti-phospho-RPS6 (S240/244) was from Cell Signaling. The horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG was from GE Healthcare. Plasmids Tet-pLKO-puro (#21915), psPAX2 (#12260), pMD2.G (#12259) were from Addgene. Titanium dioxide (TiO2) beads were from GL Sciences Inc. (Tokyo, Japan). Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (10 kD) was from Millipore.
Zhang X., Ma D., Caruso M., Lewis M., Qi Y, & Yi Z. (2014). Quantitative Phosphoproteomics Reveals Novel Phosphorylation Events in Insulin Signaling Regulated by Protein Phosphatase 1 Regulatory Subunit 12A. Journal of proteomics, 109, 63-75.
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