Total rps6

Manufactured by Cell Signaling Technology

Sourced in United States

Total RPS6 is a laboratory reagent used to quantify the expression levels of the ribosomal protein S6 (RPS6) in biological samples. RPS6 is a key component of the 40S ribosomal subunit and is involved in the regulation of protein synthesis.

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6 protocols using total 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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Examination of CIGB-325 on Virus-Induced RPS6 Phosphorylation

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MDBK cells were plated on 24-well cell culture plates at 260,000 cells per well and incubated overnight at 37 °C and 5% CO₂. Once cell monolayers were established, 14,000 TCID₅₀ of virus in 200 µL was added to each well of the plate (MOI = 0.01). After 1 h of incubation, final volume was completed up to 1 mL of serum-free DMEM and incubated. After 24 h post-infection, cells were treated with CIGB-325 (30 μM) or vehicle (PBS) in serum-free DMEM for 45 min at 37 °C in 5% CO₂. Subsequently, the culture medium was withdrawn and the cells were washed with PBS, and Western blot was carried out as described (2.7). Primary antibodies against phospho-RPS6 (S235/236) and total RPS6 (Cell Signaling Technology, Danvers, MA, USA) were used, and detection was performed with peroxidase-conjugated anti-rabbit IgG 1:5000 (Sigma, St. Louis, MO, USA).

Ramón A.C., Pérez G.V., Caballero E., Rosales M., Aguilar D., Vázquez-Blomquist D., Ramos Y., Rodríguez-Ulloa A., Falcón V., Rodríguez-Moltó M.P., Yang K., Perera Y, & Perea S.E. (2022). Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection. Viruses, 14(3), 552.

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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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Western Blot Analysis of Cellular Proteins

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Cells were lysed in cell lysis buffer (20 mM Tris, pH 7.5, 135 mM NaCl, 5% [v/v] glycerol, 50 mM NaF, 0.1% [v/v] Triton X-100, plus protease inhibitors), centrifuged and protein quantified using Bradford reagent (Sigma-Aldrich). Samples were made up in NuPAGE LDS sample buffer (Life Technologies). Western blotting was performed as previously described [28 (link)]. Blots shown are representative of 3 independent experiments. Anti-β-actin (#4967), phospho-TSC2 S1387 (#5584), total TSC2 (#3990), IRE1α (3294S), phospho-S6K1 T389 (#9205), total S6K1 (#9202), phospho-rpS6 S235/236 (#2211), total rpS6 (#2217), phospho-4E-BP1 S65 (#9451), total 4E-BP1 (#9644), phospho-ACC S79 (#3661), total ACC (#3676), PARP (#9542), caspase 3 (#9662), ATF4 (#11815), phospho-FOXO3a (#9466S), total FOXO3a (#9467), phospho-PRAS40 T246 (#2997), total PRAS40 (#2691) and phospho-Bad S136 (#4366) antibodies were from Cell Signaling Technology (Danvers, MA, USA). GADD34 antibody (10449-1-AP) was from Proteintech (Manchester, UK).

Dunlop E.A., Johnson C.E., Wiltshire M., Errington R.J, & Tee A.R. (2017). Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells. Oncotarget, 8(30), 48711-48724.

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Western Blot Analysis of Protein Expression

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Proteins were extracted using Lysis Buffer (Cell Signaling Technology) supplemented with Complete, EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics). Protein samples were separated by SDS-PAGE and subsequently transferred to PVDF membranes, which were blocked in 5% BSA and incubated with primary antibodies against V5 (Life Technologies Cat. No. R960–25), GAPDH (Santa Cruz Cat. No. sc-137179), HSP90 (Abcam Cat. No. ab13492), Vinculin (Abcam Cat. No. 18058), pRPS6 (Cell Signaling Technology Cat. No. 2215), p-AKT (Cell Signaling Technology Cat. No. 9271), p-4EBP1 (Cell Signaling Technology Cat. No. 2855), total RPS6 (Cell Signaling Technology Cat. No. 2217), total AKT (Cell Signaling Technology Cat. No. 9272), total 4EBP1 (Cell Signaling Technology Cat. No. 9644), ALK (Cell Signaling Technology Cat. No. 3633), PTEN (Cell Signaling Technology Cat. No. 9552), ERBB4 (Cell Signaling Technology, Cat. No. 4795), NRG1 (Abcam Cat. No. ab180808), BCL2L1 (Cell Signaling Cat. No. 2762), and BCL2L2 (Sigma Cat. No. SAB4502627). Membranes were washed in TBS-T and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Signal was detected by enhanced chemi-luminescence (ThermoFisher Scientific).

Iniguez A.B., Alexe G., Wang E.J., Roti G., Patel S., Chen L., Kitara S., Conway A., Robichaud A.L., Stolte B., Bandopadhayay P., Goodale A., Pantel S., Lee Y., Cheff D.M., Hall M.D., Guha R., Davis M.I., Menard M., Nasholm N., Weiss W.A., Qi J., Beroukhim R., Piccioni F., Johannessen C, & Stegmaier K. (2018). Resistance to epigenetic-targeted therapy engenders tumor cell vulnerabilities associated with enhancer remodeling. Cancer cell, 34(6), 922-938.e7.

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Protein Expression Analysis in Spheroid and Colon Tissue

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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 (20 (link)). 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; Thr308, 2965, 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. β-actin (5125, Cell Signaling Technology) was used as a loading control at a ratio of 1:1000.

Fricke S.L., Payne S.N., Favreau P.F., Kratz J.D., Pasch C.A., Foley T.M., Yueh A.E., Van De Hey D.R., Depke M.G., Korkos D.P., Sha G.C., DeStefanis R.A., Clipson L., Burkard M.E., Lemmon K.K., Parsons B.M., Kenny P.A., Matkowskyj K.A., Newton M.A., Skala M.C, & Deming D.A. (2018). MTORC1/2 inhibition as a therapeutic strategy for PIK3CA mutant cancers. Molecular cancer therapeutics, 18(2), 346-355.

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