Phospho rps6

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-RPS6 is a lab equipment product from Cell Signaling Technology. It is used to detect the phosphorylation of the ribosomal protein S6 (RPS6), a downstream target of the PI3K/Akt/mTOR signaling pathway.

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Lab products found in correlation

Close spelling variants of this product

Phospho-RPS6 (Ser235/236) (10 citations) Anti-phospho-RPS6 (7 citations) Anti-rpS6 phospho-Ser235/236 (3 citations) Anti-phospho-rpS6 (ser240/244 (3 citations) Phospho-rpS6 (S235/ 236), (3 citations) Phospho-RPS6 (Ser 235/236) (D57.2.2.E; (2 citations) Phospho-RPS6 (S235/236) and total RPS6 (2 citations) Phospho-RPS6 (Ser 240/244) (D68F8; (2 citations)

12 protocols using phospho rps6

Immunoblotting of Phosphorylated Proteins

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Tissue samples were collected and flash frozen. After 24 h, protein was extracted and immunoblotting performed as previously described.^{43 (link)} The membranes were blocked with 5% non-fat dry milk for 1 h and then probed with primary antibodies against phospho RPS6 (Ser235/236, 4858, Cell Signaling Technology), phospho AKT (Ser473, 4060, Cell Signaling Technology), phospho 4EBP1 (Thr37/46, 2855, Cell Signaling Technology), total RPS6 (2217, Cell Signaling Technology), total AKT (4691, Cell Signaling Technology) or total 4EBP1(9644, Cell Signaling Technology) in bovine serum albumin at 1:1000. Anti-GAPDH antibody (8884, Cell Signaling Technology) was used as a loading control at a ratio of 1:5000.

Payne S.N., Maher M.E., Tran N.H., Van De Hey D.R., Foley T.M., Yueh A.E., Leystra A.A., Pasch C.A., Jeffrey J.J., Clipson L., Matkowskyj K.A, & Deming D.A. (2015). PIK3CA mutations can initiate pancreatic tumorigenesis and are targetable with PI3K inhibitors. Oncogenesis, 4(10), e169-.

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Salternamide A Biochemical Assay

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Salternamide A (SA, Figure 1A) was dissolved in 100% DMSO and stored at −20 °C for subsequent analysis. Cobalt (II) chloride (CoCl₂) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for HIF-1α, Akt, phospho-Akt (Thr³⁰⁸), PI3K, phospho-PI3K (Tyr^458/199), RPS6, phospho-RPS6 (Ser^235/236), p70S6K1, phospho-p70S6K1 (Thr³⁸⁹), phospho-STAT3 (Tyr⁷⁰⁵), mTOR, phospho-mTOR (Ser²⁴⁴⁸), 4E-BP1, phospho-4E-BP1 (Thr^37/46), eIF4E, phospho-eIF4E (Ser²⁰⁹), phospho-CDC2 (Thr¹⁶¹), CDC25C, phospho-CDC25C (Ser²¹⁶), Chk1, phospho-Chk1 (Ser³⁴⁵), phospho-Chk2 (Thr¹⁶⁸), caspase-3, caspase-8, caspase-9, cleaved caspase-3, cleaved caspase-8, and LC3B were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for ERK 1/2, phospho-ERK 1/2 (Thr²⁰²/Tyr²⁰⁴), STAT3, CDC2, cyclin B1, cyclin A, Bcl-2, and Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for VHL, PARP, cleaved PARP, and Bim were purchased from BD Pharmingen™ (BD Biosciences, San Jose, CA, USA). Hsp90 antibody was purchased from Stressgen Bioreagents (Ann Arbor, MI, USA).

Bach D.H., Kim S.H., Hong J.Y., Park H.J., Oh D.C, & Lee S.K. (2015). Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells. Marine Drugs, 13(11), 6962-6976.

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Immunoblotting of mTOR Pathway Proteins

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The phospho and total antibodies used for immunoblotting were purchased from Cell Signaling (Beverly, CA): phospho‐Akt (Ser³⁰⁸; 1:1000), phospho‐mTOR (Ser²⁴⁴⁸; 1:500), phospho‐S6K1 (Thr³⁸⁹;1: 500), phospho‐4E‐BP1 (Thr^37/46; 1:1000), and phospho‐rpS6 (Ser^240/244; 1:250). Total protein was detected for Akt (1:1000), mTOR (1:500), S6K1 (1:500), 4E‐BP1 (1:1000), and rpS6 (1:250). Anti‐rabbit IgG HRP‐conjugated secondary antibody was purchased from Amersham Bioscience (1:2000).

Walker D.K., Drummond M.J., Dickinson J.M., Borack M.S., Jennings K., Volpi E, & Rasmussen B.B. (2014). Insulin increases mRNA abundance of the amino acid transporter SLC7A5/LAT1 via an mTORC1‐dependent mechanism in skeletal muscle cells. Physiological Reports, 2(3), e00238.

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Comprehensive Cell Lysis and Immunoblotting

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Cells were lyzed using 0.20% NP40, 50 mM Tris pH 7.5, 5% Glycerol, 1.5 mM MgCl₂, 100 mM NaCl lysis buffer containing Phosphatase Inhibitor Cocktail 2 (Sigma, P5726) and cOmplete Protease Inhibitor Cocktail (Roche, 11873580001). Lysates were resolved by SDS-PAGE and incubated with primary antibodies. Antibodies used were against actin (A5441), CAMKK2 (HPA017389) (both Sigma), ERK1/2 (Sigma, M5670) and phospho-AKT (Ser473)(#9271), AKT (#9272), ALK(#3633), phospho-p90RSK (Ser380)(#12032), RSK1/2/3 (#9355), RSK1(#9333), RSK2 (#5528), phospho-RPS6 (Ser235/236)(#4858), RPS6 (#2217), phospho-ERK1/2 (Thr202/Tyr204)(#4267), phospho-FAK1 (Tyr397)(#8556), FAK1 (#13009), phospho-IGF1R (Tyr1131)(#3021), IGF1R (#9750), cleaved Caspase 3 (#9661), PARP-1 (#9542), AMPK1 (#2795), pAMPKα (Thr172)(#2535), FER (#4268), phospho-YB1 (Ser102)(#2900), and YB1 (#4202) were from Cell Signaling. Secondary antibodies were HRP-conjugated α-rabbit or α-mouse (GE Healthcare).

Kuenzi B.M., Remsing Rix L.L., Stewart P.A., Fang B., Kinose F., Bryant A.T., Boyle T.A., Koomen J.M., Haura E.B, & Rix U. (2017). Polypharmacology-based ceritinib repurposing using integrated functional proteomics. Nature chemical biology, 13(12), 1222-1231.

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Western Blot Antibody Panel for Cell Signaling

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Primary antibodies against GFP (Takara Bio, 632380, AB_10013427; 1:5000), G-6-PDH (Sigma-Aldrich, A9521, AB_258454; 1:5000), Adh1 (Millipore, 126745, AB_564196; 1:200000), RPS6 (Cell Signaling Technology, 2217, AB_331355; 1:1000), phospho-RPS6 (Ser^235-236) (Cell Signaling Technology, 4856, AB_2181037; 1:1000), RPS6KB (Cell Signaling Technology, 2708, AB_390722; 1:1000), phospho-RPS6KB (Thr³⁸⁹) (Cell Signaling Technology, 9205, AB_330944; 1:1000), EIF4EBP1 (Cell Signaling Technology, 9452, AB_331692; 1:1000), phospho-EIF4EBP1 (Thr^37/46) (Cell Signaling Technology, 2855, AB_560835; 1:1000), phospho-EIF4EBP1 (Ser⁶⁵) (Cell Signaling Technology, 9451, AB_330947; 1:1000), AKT1 (Cell Signaling Technology, 4691, AB_915783; 1:1000), phospho-AKT(Ser⁴⁷³) (Cell Signaling Technology, 4060, AB_2315049; 1:1000), phospho-AKT(Thr³⁰⁸) (Cell Signaling Technology, 13038, AB_2629447; 1:1000), ULK1 (Cell Signaling Technology, 8359, AB_11178668; 1:1000), phospho-ULK1(Ser⁷⁵⁷) (Cell Signaling Technology, 6888, AB_10829226; 1:1000), MAP1LC3 AB (Cell Signaling Technology, 12741, AB_2617131; 1:1000), β-actin (Cell Signaling Technology, 4967, AB_330288; 1:1000) were used.

Nicastro R., Gaillard H., Zarzuela L., Péli-Gulli M.P., Fernández-García E., Tomé M., García-Rodríguez N., Durán R.V., De Virgilio C, & Wellinger R.E. (2022). Manganese is a physiologically relevant TORC1 activator in yeast and mammals. eLife, 11, e80497.

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Immunoblot Analysis of Phosphorylated RPS6

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Keratinocytes were grown in culture plates and directly lysed using 1X SDS sample buffer containing 1% SDS, 50 mM TrisHCl pH 6.8, 10% glycerol, 50 mM DTT and Bromophenol blue. Samples were boiled and centrifuged for 1 minute at 16,000 × g. Samples were separated on mini-PROTEAN 4–20% TGX gels (Biorad) and blotted on PVDF membranes. Membranes were blocked with Protein Free Blocking Buffer (Thermo scientific) and Immunostained overnight with phospho-RPS6 (5364, Cell Signaling Technology) and alpha-tubulin (T6074, Sigma-Aldrich) antibodies. Staining with secondary antibodies (LiCor) was performed for one hour and membranes where scanned using the Odyssey CLx imaging system from LiCor.

Gerlach J.P., van Buggenum J.A., Tanis S.E., Hogeweg M., Heuts B.M., Muraro M.J., Elze L., Rivello F., Rakszewska A., van Oudenaarden A., Huck W.T., Stunnenberg H.G, & Mulder K.W. (2019). Combined quantification of intracellular (phospho-)proteins and transcriptomics from fixed single cells. Scientific Reports, 9, 1469.

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Pharmacological Inhibition of mTOR

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RAD001 (No 07741; Sigma-Aldrich Co., St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO). All other chemicals, unless otherwise stated, were from Sigma-Aldrich Co. Antibodies against mTOR, phospho-mTOR (Ser2448), phospho-mTOR (Ser2481), AKT, phospho-AKT (Ser473), rpS6, phospho-rpS6, 4EBP1 and phospho-4EBP1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against enhanced green fluorescent protein (EGFP) and β-actin were from Sigma-Aldrich Co.

Tsai T.F., Lin J.F., Chou K.Y., Lin Y.C., Chen H.E, & Hwang T.I. (2018). miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells. OncoTargets and therapy, 11, 239-252.

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Immunoblotting Analysis of Signaling Pathways

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Spheroid and colon tissue samples were collected and flash frozen. After 24 hours, protein was extracted and immunoblotting performed as previously described (50). The membranes were blocked with 5% non-fat dry milk for one hour and then probed with primary antibodies against phospho RPS6 (Ser235/236, #4858, Cell Signaling Technology), phospho AKT (Ser473, #4060, Cell Signaling Technology), phospho 4EBP1 (Thr37/46, #2855, Cell Signaling Technology), phospho EEF2 (Thr 56, #2331, Cell Signaling Technology), total RPS6 (#2217, Cell Signaling Technology), total AKT (#4691, Cell Signaling Technology), total 4EBP1 (9644, Cell Signaling Technology)), or total eEF2 (#2332, Cell Signaling Technology) in bovine serum albumin at 1:1000. Anti-GAPDH (#8884, Cell Signaling Technology) or β-actin (#5125, Cell Signaling Technology) antibodies were used as a loading control at a ratio of 1:1000.

Foley T.M., Payne S.N., Pasch C.A., Yueh A.E., Van De Hey D.R., Korkos D.P., Clipson L., Maher M.E., Matkowskyj K.A., Newton M.A, & Deming D.A. (2017). Dual PI3K/mTOR Inhibition in Colorectal Cancers with APC and PIK3CA Mutations. Molecular cancer research : MCR, 15(3), 317-327.

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Signaling Pathway Regulation in Cells

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Fluocinolone acetonide (FA), croton oil (CO), Wortmannin (WM), AZD8055 (AZD), NVP-BEZ235 (NVP), MK-2206 (MK), and 5-bromo-2′-deoxyuridine (BrdU), were from Sigma Aldrich (St. Louis, MO), LY294002 (LY) was from LC Laboratories (Woburn, MA). TNF-α and cytoplasmic and nuclear fractionation kit were from Thermo Fisher Scientific (Waltham, MA).
We used antibodies to GR (sc-8992, RRID:AB_2155784), RelA/p65 (sc-8008, RRID:AB_628017), phospho-RelA/p65 (Ser536) (sc-136548, RRID:AB_10610391), IκB (sc-1643, RRID:AB_627772), phospho-IκB (Ser32) (sc-8404, RRID:AB_627773), FKBP51 (sc-271547, RRID:AB_10649040) (Santa Cruz Biotechnology, Dallas, TX); GAPDH (G9545, RRID:AB_796208, Sigma Aldrich); phospho-GR (Ser211) (4161, RRID:AB_2155797), phospho-rpS6 (Ser235/236) (4856S, RRID:AB_2181037), phospho-4E-BP1 (Thr37/46) (9459L, RRID:AB_2262165), phospho-P70S6K (Thr389) (9234, RRID:AB_2269803), phospho-AKT (Thr308 and Ser473) (9266S, RRID:AB_659801 and 9271, RRID:AB_329825), lamin B (12586, RRID:AB_2650517) and tubulin (2148, RRID:AB_2288042) (Cell Signaling, San Jose, CA), REDD1 (10638-1-AP, RRID,AB_2245711, Proteintech Group, Rosemont, IL).

Agarwal S., Mirzoeva S., Readhead B., Dudley J.T, & Budunova I. (2019). PI3K inhibitors protect against glucocorticoid-induced skin atrophy. EBioMedicine, 41, 526-537.

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Antibody Characterization and Validation

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All antibodies were ordered without any carrier proteins that could interfere with antibody functionalization. EGFR (Ab231, Abcam), NOTCH1 (AF5317, RnD systems), JAG1 (AF1277, RnD systems), KLK6 (AF2008, RnD systems), phospho-FAK (AF4528, RnD systems), phospho-RPS6 (5364, Cell Signaling Technology), ITGA6 (Produced and purified in house from hybridoma P5G10, DSHB), TGM1 (Produced and purified in house from BC1 hybridoma, a kind gift from Prof. Robert Rice), alpha-tubulin (T6074, Sigma-Aldrich).

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