Phospho s6 ser235 236

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-S6 (Ser235/236) is a lab equipment product that detects and quantifies the phosphorylation of ribosomal protein S6 at serine residues 235 and 236. This product can be used to analyze cellular signaling pathways involving the mTOR and S6 kinase signaling cascades.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Phospho-S6 (110 citations) Phospho-S6 Ribosomal Protein (Ser235/236) (34 citations) Rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (10 citations) Rabbit anti-phospho-S6 (Ser235/236) (9 citations) Anti-Phospho-S6 Ribosomal protein (Ser235/236) antibody (4 citations) PathScan Phospho-S6 Ribosomal Protein (Ser235/236) Sandwich ELISA Kit (4 citations) Anti-phospho ribosomal protein S6 (S6) (Ser235/236), (3 citations) Phospho-S6 Ribosomal Protein (Ser235/236) Antibody (3 citations) Anti-human phospho-S6 ribosomal protein (Ser235/236) (2 citations) Phospho-S6 (ser235/236) and S6 rabbit monoclonal antibodies (2 citations)

32 protocols using phospho s6 ser235 236

Western Blot Analysis of Protein Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total soluble protein was extracted from the whole heart tissue after completion of experimental protocol as described above. The extraction buffer consisted of 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM PMSF, 1 μg/mL pepstatin, 05 μg/mL leupeptin, 2 mM NaF, 0.2 mM Na₃VO₄, 5 mM β-mecaptoethanol. The homogenate was centrifuged at 14,000g for 15 min at 4 °C and the supernatant was recovered. Protein (50 μg) from each sample was separated by SDS-PAGE and transferred onto nitrocellulose membrane [10 (link)–12 (link)]. The membrane was incubated overnight with primary antibody at a dilution 1:1000 for each of the respective proteins (i.e., phosphotyrosine⁷⁰⁵-STAT3, STAT3, phospho-Ser^235/236-S6, S6, phosphor Ser⁴⁷³-AKT and AKT from Cell Signaling, MA; and GAPDH from Santa Cruz Biotechnology, TX). The membrane was washed and incubated with horseradish peroxidase conjugated secondary antibody (1:2000 dilutions) for 1 h. The blots were developed using a chemiluminescent system (ECL Plus; Amersham Biosciences).

Das A., Salloum F.N., Filippone S.M., Durrant D.E., Rokosh G., Bolli R, & Kukreja R.C. (2015). Inhibition of mammalian target of rapamycin protects against reperfusion injury in diabetic heart through STAT3 signaling. Basic research in cardiology, 110(3), 31.

+ Open protocol

+ Expand

Protein Extraction and Western Blotting

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in radio-immunoprecipitation assay buffer (10 mM HEPES buffer, pH 7.6, 42 mM KCl, 5 mM MgCl₂, 1% SDS, 1 mM phenylmethylsulfonylfluoride, 1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM ethylene glycol tetraacetic acid, 1 mM dithiothreitol, 1.5 μM pepstatin, 2 μM leupeptin, and 0.7 μM aprotinin). Protein concentration was determined using the DCTM protein assay (Bio-Rad, Hercules, CA, USA). Thirty to fifty micrograms of protein was applied for polyacrylamide gel electrophoresis. The primary antibodies against HMGB1 (1 : 500), RIPK3 (1 : 1000), Akt1 (1 : 500), Akt2 (1 : 500), Akt3 (1 : 500), Akt (1 : 500), mTOR (1 : 500), phospho (Ser-473)-Akt (1 : 500), phospho (Ser308)-Akt (1 : 500), phospho (Ser9)-GSK-3β (1 : 1000), phospho (Ser235/236)-S6 (1 : 1000), and phospho (ser2448)-mTOR (1 : 1000) were obtained from Cell Signaling. Horseradish peroxidase conjugated secondary antibodies (1 : 10 000) were used for ECL-plus (GE Healthcare, Pittsburgh, PA, USA) detection. The results were normalized to β-actin (1 : 5000).

Liu Q., Qiu J., Liang M., Golinski J., van Leyen K., Jung J.E., You Z., Lo E.H., Degterev A, & Whalen M.J. (2014). Akt and mTOR mediate programmed necrosis in neurons. Cell Death & Disease, 5(2), e1084-.

+ Open protocol

+ Expand

Anticancer Drug Screening in Cell Lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

MiaPaCa2, MDA-MB231, Panc1, AsPC1, and HEK293T cell lines were obtained from American Type Culture Collection (ATCC) and mycoplasma-free. These cells were cultured in Dulbecco’s minimal essential medium (DMEM) from Nacalai (California, USA) supplemented with 10% v/v FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) from Hyclone (IL, USA). Antibodies for GAPDH (14C10, #2118), phospho-AKT Ser473 (#9271), phospho-ERK Thr202/Tyr204 (#9101), phospho-S6 Ser235/236 (#2211), YAP (#4912), phospho-YAP Ser127 (#4911), and phospho-LATS1 Ser909 (#9157) were from Cell Signaling Technology (MA, USA). Antibody for TAZ (#HPA007415) was from Sigma-Aldrich (MO, USA). Gemcitabine-HCl (#S1149) was obtained from Selleck Chemicals (TX, USA), while doxorubicin-HCl (#D-4000) from LC Laboratories (MA, USA). PD184352, Triciribine and Rapamycin were obtained from Sigma-Aldrich (MO, USA). The Cell viability was assayed by colorimetric based CellTiter 96^® AQueous One Solution Cell Proliferation kit (#G3581, Promega), per manufacture’s protocol.

Chai T.F., Manu K.A., Casey P.J, & Wang M. (2020). Isoprenylcysteine carboxylmethyltransferase is required for the impact of mutant KRAS on TAZ protein level and cancer cell self-renewal. Oncogene, 39(31), 5373-5389.

+ Open protocol

+ Expand

Molecular Response to Taselisib and Letrozole

Check if the same lab product or an alternative is used in the 5 most similar protocols

Taselisib, also called GDC-0032, was generated at Genentech, Inc. (South San Francisco, CA). Letrozole was obtained from US Biological. Antibodies used include phospho-AKT^Ser473, AKT, phospho-PRAS40^Thr246, phospho-S6^Ser235/236, phospho-S6^Ser240/242, S6, phospho-ERK^{Thr202/Tyr204}, ERK, phospho-ERα^Ser118, phospho-ERα^Ser167, cleaved PARP, p110α, phospho-p70S6K^Thr389, PR, cyclin E, phospho-mTOR^Ser2448, IGF1R, BRCA1, c-Myc, CAV1, HER2 and cyclin D1 obtained from Cell Signaling (Danvers, MA). Antibodies for ERα and ERβ were obtained from Santa Cruz biotechnology (Santa Cruz, CA) and a βActin antibody was obtained from Sigma (St. Louis, MO).

Hoeflich K.P., Guan J., Edgar K.A., O'Brien C., Savage H., Wilson T.R., Neve R.M., Friedman L.S, & Wallin J.J. (2016). The PI3K inhibitor taselisib overcomes letrozole resistance in a breast cancer model expressing aromatase. Genes & Cancer, 7(3-4), 73-85.

+ Open protocol

+ Expand

Plantaris Muscle Protein Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Plantaris muscles were homogenized in ice-cold buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl₂, 1 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, 2 mM PMSF, aprotinin (10 μg/mL), and leupeptin (10 μg/mL). The homogenates were rotated end-over-end at 4 °C for 60 min and then centrifuged at 10,000× g for 10 min at 4 °C. Aliquots of supernatants were used for immunoblot analysis. Briefly, supernatants were electrophoretically separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with primary antibodies, followed by incubation for 60 min with appropriate HRP-conjugated second antibodies. Primary antibodies against mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Thr308), S6, and phospho-S6 (Ser235/236), cAMP response element-binding protein (CREB), and phospho-CREB (Ser133) were obtained from Cell Signaling Technology (Beverly, MA). Immunoreactive bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Japan, Hino, Japan), and quantified using NIH Image software.

Koshinaka K., Honda A., Iizumi R., Miyazawa Y., Kawanaka K, & Sato A. (2021). Egg White Protein Feeding Facilitates Skeletal Muscle Gain in Young Rats with/without Clenbuterol Treatment. Nutrients, 13(6), 2042.

+ Open protocol

+ Expand

Rapamycin and IGF-1 Signaling Pathway Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Rapamycin (LC Laboratories, Woburn, MA) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 μg/ml stock solution and stored at −20°C. IGF-1 (PeproTech, Rocky Hill, NJ) was rehydrated in 0.1 M acetic acid to prepare a 10 μg/ml stock solution and stored at −80°C. Shrimp alkaline phosphatase (1,000 units/ml, New England BioLabs, Ipswich, MA). Enhanced chemiluminescence solution was from Pierce (Rockford, IL). Antibodies included those against mTOR, S6K1, Akt, S6, HA (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-S6K1 (Thr389), phospho-Akt (Ser473), phospho-S6 (Ser/235/236), 4E-BP1 (Cell Signaling, Beverly, MA), raptor, rictor (Bethyl Laboratories, Montgomery, TX), mLST8 (GenWay Biotech, San Diego, CA), mSin1 (for Western blotting, K87 [13 (link)]; for immunoprecipitation, sc-48588, Santa Cruz Biotechnology), β-tubulin, FLAG (Sigma, St. Louis, MO); goat anti-rabbit IgG-horseradish peroxidase (HRP), goat anti-mouse IgG-HRP, rabbit anti-goat IgG-HRP, and goat anti-chicken IgG-HRP (Pierce). All other chemicals were purchased from Sigma.

Luo Y., Liu L., Wu Y., Singh K., Su B., Zhang N., Liu X., Shen Y, & Huang S. (2015). Rapamycin inhibits mSin1 phosphorylation independently of mTORC1 and mTORC2. Oncotarget, 6(6), 4286-4298.

+ Open protocol

+ Expand

Comparative Protein Quantification in Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Fifty Microgram/Microliter of protein was separated in a 10% SDS-PAGE gel under denaturing conditions, transferred to PVDF membranes and probed with: phospho-AMPKα (Thr172), AMPKα, phospho-PKB (Thr308), PKB/Akt, phospho-S6 (Ser235/236) or S6 (all from Cell Signaling Technology). The membranes were revealed utilizing a chemiluminescence assay and the amount of protein was analyzed utilizing Quantity One software (Bio-Rad Laboratories). Actin, detected with an antibody (Sigma-Aldrich Corporation), was used as loading control.

Phillips-Farfán B.V., Rubio Osornio M.D., Custodio Ramírez V., Paz Tres C, & Carvajal Aguilera K.G. (2015). Caloric restriction protects against electrical kindling of the amygdala by inhibiting the mTOR signaling pathway. Frontiers in Cellular Neuroscience, 9, 90.

+ Open protocol

+ Expand

Multiparameter Flow Cytometry Immunophenotyping

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were labelled with allophycocyanin (APC) CD11c (HL3), BV421 CD11c (HL3),
FITC CD80 (16-10A1), CD86 PE (GL1), APC CD40 (1C10), PerCP-eFluor 710 CD40
(1C10), PE-Cy7 CD19 (1D3), PerCP-Cy5.5 (H1.2F3), APC TCRβ (H57-597),
phycoerythrin (PE) TCRβ (H57-597), FITC CD3 (145-2c11), APC-eFluor 780
major histocompatibility complex II (M5/114.15.2), BV605 CD45.1 (A20), BV786
CD45.2 (104) and APC IFNγ (XMG1.2) purchased from eBioscience or BD
Pharmingen. pS6 analysis used phospho-S6 Ser 235/236 (Cell Signaling
Technologies) and secondary was PE-conjugated donkey anti-rabbit immunoglobulin
G (Jackson ImmunoResearch). 2-NBDG (Life Technologies) was added to cells at
35 μM for 1 h prior to analysis. Live cells were
gated by forward scatter (FSC-A) and side scatter (SSC-A) analysis. Single cells
were selected by FSC-A and FSC-W analysis. For intracellular staining, cells
were then fixed and permeabilized using Cytofix/Cytoperm reagent (BD
Pharmingen). For cytokine analysis, endocytosis was blocked using golgi plug (BD
Pharmingen) for 4 h. Gating strategies for all flow cytometry
analysis are outlined in Supplementary
Figs 5 and 6. Data were acquired on a FACSCanto or LSRFortessa (Becton
Dickinson) and analysed using the FlowJo software (TreeStar).

Lawless S.J., Kedia-Mehta N., Walls J.F., McGarrigle R., Convery O., Sinclair L.V., Navarro M.N., Murray J, & Finlay D.K. (2017). Glucose represses dendritic cell-induced T cell responses. Nature Communications, 8, 15620.

+ Open protocol

+ Expand

Immunoblotting Analysis of SNAT1 and mTOR Pathway

Check if the same lab product or an alternative is used in the 5 most similar protocols

Tissues or cultured cells were solubilized in lysis buffer containing 1% Triton X-100. Samples were subjected to SDS-PAGE, followed by electrophoretic transfer to polyvinylidene fluoride membranes and subsequent immunoblotting. Immunoblotting was performed using antibodies targeting SNAT1 (kindly provided by Jeffrey D. Erickson), phospho-p70 S6 kinase (Thr389) (#9234 Cell Signaling Technology), p70 S6 kinase (#2708 Cell Signaling Technology), phospho-mTOR (Ser2448) (#2971 Cell Signaling Technology), phospho-S6 (Ser235/236) (#4858 Cell Signaling Technology), phospho-Akt (Ser473) (#4060 Cell Signaling Technology), GAPDH (sc-25778 Santa Cruz Biotechnology, Inc.), β-tubulin (T4026 Sigma-Aldrich), and β-actin (sc-4778 Santa Cruz Biotechnology, Inc.). Primary antibodies were diluted 2000-fold with blocking solution (5% skim milk in TBST [137 mM NaCl, 0.05% Tween 20, 20 mM Tris–HCl buffer, pH 7.5]).

Yamada D., Kawabe K., Tosa I., Tsukamoto S., Nakazato R., Kou M., Fujikawa K., Nakamura S., Ono M., Oohashi T., Kaneko M., Go S., Hinoi E., Yoneda Y, & Takarada T. (2019). Inhibition of the glutamine transporter SNAT1 confers neuroprotection in mice by modulating the mTOR-autophagy system. Communications Biology, 2, 346.

+ Open protocol

+ Expand

Pancreatic Immunohistochemistry and Islet Morphometry

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Pancreatic tissue was fixed in 10% neutral buffered formalin (Surgipath Leica, Buffalo Grove, IL) and embedded in paraffin for sectioning and processing as previously described (Albury et al. 2015 (link)). Slides were stained for hematoxylin and eosin (Surgipath) or immunohistochemistry using the Polymer Refine Detection reagents (Leica) on the Bond-Max immunostainer (Leica). Antigen retrieval was optimized using sodium citrate (pH 6) or EDTA (pH 9) buffers (Leica). The following antibodies were used: phospho-Akt Ser473 (GeneTex, Irvine, CA), also phospho-mTor Ser2448, phospho-S6 Ser235/236, glucagon, and insulin (all from Cell Signaling Technology, Danvers, MA). All slides processed on the immunostainer were run with a negative control, which was treated with antibody diluent instead of the primary antibody, to ensure antibody specificity. Images were taken using a Leica DM 2000 microscope with 5X, 10X, or 40X objectives. Islet size was measured in the whole pancreas of three mice per genotype. The mice selected had no significant lesions (NSL) at the time of necropsy, as described by a pathologist. The pancreas was sectioned into 5μm sections and every 25^th section was H&E stained. A total of 50 islets from three sections were analyzed per mouse. Islet diameter was determined using measuring tools available on an Axio Imaging System (Zeiss, Oberkochen, Germany).

Albury-Warren T.M., Pandey V., Spinel L.P., Masternak M.M, & Altomare D.A. (2015). Prediabetes Linked to Excess Glucagon in Transgenic Mice with Pancreatic Active AKT1. The Journal of endocrinology, 228(1), 49-59.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!