Phospho s6 s235 236

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-S6 S235/236 is a lab equipment product that detects the phosphorylation of the ribosomal protein S6 at serine residues 235 and 236. This product is used to measure the activation of the mTOR signaling pathway.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Phospho-S6 Ribosomal Protein (S235/236, Rabbit anti-phospho-S6 S235/236 Anti-phospho-S6 S235/236 Phospho-S6

20 protocols using phospho s6 s235 236

Immunoblot Analysis of Cell Signaling

Cited 1 time

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

All immunoblot analyses were performed as previously described [59 (link)]. Antibodies were obtained from the following sources: antibodies to phospho-T389-S6K1, phospho-S473-AKT, phospho-S235/236-S6, phospho-S65-4E-BP1, 4E-BP1 S6K1, AKT, S6, GAPDH, RICTOR, RAPTOR, DEPTOR, PRAS40, mLST8, mSIN1 were obtained from Cell Signaling Technology. Antibodies to myc (9E10) and FLAG (M2) were obtained from Sigma.

Ghosh A.P., Marshall C.B., Coric T., Shim E.H., Kirkman R., Ballestas M.E., Ikura M., Bjornsti M.A, & Sudarshan S. (2015). Point mutations of the mTOR-RHEB pathway in renal cell carcinoma. Oncotarget, 6(20), 17895-17910.

+ Open protocol

+ Expand

Protein Extraction and Immunoblotting

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

Cell proteins were extracted with buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton-X 100), supplemented with protease and phosphatase inhibitors (GeneDepot, Barker, TX). For each sample, 50 μg of protein was resolved on SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was performed using the following antibodies: INPP4B 1:1000 (Santa Cruz), FLAG M2 (1:5000) (Sigma-Aldrich), β-Tubulin (1:2000) (Millipore, Billerica, MA), total Akt (1:1000), phospho- S473 Akt (1:1000), COX-2 (1:1000), phospho-S235/236 S6 (1:1000), total S6 (1:1000), survivn/BIRC5 (1:1000), pan phospho-PKC (1:1000) (Cell Signaling Technology, Beverly, MA), PAK6 (1:400) (R&D Systems, Minneapolis, MN). Luminescent signal was captured on a Gel Logic 2000 imaging system with Carestream Molecular Imaging software (Carestream, Rochester, NY).

Hodgson M.C., Deryugina E.I., Suarez E., Lopez S.M., Lin D., Xue H., Gorlov I.P., Wang Y, & Agoulnik I.U. (2014). INPP4B suppresses prostate cancer cell invasion. Cell Communication and Signaling : CCS, 12, 61.

+ Open protocol

+ Expand

Immunofluorescence Staining of DRG Neurons

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

DRGs were cultured as described above and plated in eight-well chamber slides (Nunc Lab-Tek, Thermo Fisher Scientific). Cultures were fixed in ice-cold 4% PFA in 1× PBS for 20 min and then washed with PBS three times for 5 min each time. Cells were then permeabilized with 0.025% Triton X-100 (Sigma) in PBS containing 1% BSA for 30 min. After washes, slides were blocked by 10% NGS and 1% BSA in PBS for at least 1 h. Primary antibodies were used to detect the following proteins: c-Fos (1:20; Sigma, cat. # PC05, RRID:AB_564450), phospho-S6 (S^235/236) (1:100; Cell Signaling Technology [CST], cat. # 4858, RRID:AB_916156), phospho-S6 (S^240/244) (1:800; CST, cat. # 5364, RRID:AB_10694233), S6 ribosomal protein (1:200: CST, cat. # 2217, RRID:AB_331355) and peripherin (1:1000; Novus, cat. # NBP1-05423, RRID:AB_1556333). Primary antibodies were incubated at 4°C overnight. The next day, appropriate secondary antibodies (Alexa Fluor, Invitrogen) were applied for 1 h. Following additional PBS washes, coverslips were mounted with ProLong Gold.

de la Peña J.B., Kunder N., Lou T.F., Chase R., Stanowick A., Barragan-Iglesias P., Pancrazio J.J, & Campbell Z.T. (2021). A role for translational regulation by S6 kinase and a downstream target in inflammatory pain. British journal of pharmacology, 178(23), 4675-4690.

+ Open protocol

+ Expand

Western Blotting of SCLC Proteins

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

Protein lysates from SCLC lines and murine tumors were prepared in RIPA buffer, supplemented with PhosSTOP phosphatase and cOmplete protease inhibitor cocktail (Roche #11873580001, #04906845001). Protein samples were quantified with the BCA assay. Samples were separated on 4–20% SDS-PAGE gels, transferred to PVDF membranes, and probed with antibodies against ASCL1 (BD Bioscience #556604); ACTB (Sigma #A3854); IMPDH1 (Sigma #SAB2101156); IMPDH2 (Abcam #ab131158); MAT2A (Abcam #ab77471); MYC (Cell Signaling #5605); GMPS (Cell Signaling #14602); Cleaved Caspase-3 (Cell Signaling #9664); mTOR (Cell Signaling #9862); Phospho-mTOR S2448 (Cell Signaling #9862); Phospho-4EBP1 T37/46 (Cell Signaling #9862); Phospho-p70 S6 Kinase T389 (Cell Signaling #9234); Phospho-S6 S235/236 (Cell Signaling #4858). Bands were detected with the ECL blotting system (Pierce #32106).

Huang F., Ni M., Chalishazar M.D., Huffman K.E., Kim J., Cai L., Shi X., Cai F., Zacharias L.G., Ireland A.S., Li K., Gu W., Kaushik A.K., Liu X., Gazdar A.F., Oliver T.G., Minna J.D., Hu Z, & DeBerardinis R.J. (2018). Inosine monophosphate dehydrogenase dependence in a subset of small cell lung cancers. Cell metabolism, 28(3), 369-382.e5.

+ Open protocol

+ Expand

Letrozole and Estradiol Signaling Regulation

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

Reagents used were as follows: letrozole (Sigma, L6545), 17β-estradiol (E2) (Sigma, 491187), celecoxib (Sigma, PZ0008), PGE2 (Sigma, P5640), tunicamycin (Sigma, T7765), salubrinal (Calbiochem, 324895), rapamycin (Sigma, R0395), arachidonic acid (Sigma, A9673), MTT (Sigma, M2128), Z-VAD-fmk (Sigma, V116), bafilomycin A1 (Sigma, B1793), 3-MA (Sigma, 08592), Hoechst 33342 (Sigma, B2261), acridine orange (Sigma, A6014), DMSO (Sigma, D2650). letrozole, E2, celecoxib, PGE2, tunicamycin, and salubrinal were dissolved in DMSO, while MTT, Hoechst 33342, and acridine orange were dissolved in phosphate-buffered saline (PBS).
Antibodies were obtained from the following sources: antibodies against Beclin 1, COX-2, EP-4, β-actin, Bcl-2, BAX, phospho-4EBP1 and phospho-Akt (S473) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Phospho-p70-S6K(T389), eIF2α, phospho-eIF2α, Raptor, phospho-mTOR(S2448), caspase 3, and phospho-S6(S235/236) were from Cell Signaling Inc (Beverly, MA); LC3, ATG5, protein disulfide isomerase (PDI) were from Abcam (Cambridge, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Zhou S., Zhao L., Yi T., Wei Y, & Zhao X. (2016). Menopause-induced uterine epithelium atrophy results from arachidonic acid/prostaglandin E2 axis inhibition-mediated autophagic cell death. Scientific Reports, 6, 31408.

+ Open protocol

+ Expand

Epidermal Protein Expression Analysis

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

Facial hair was removed using Nair® hair removal cream and skin around the cheek and eyelid area was dissected out in one piece, snap frozen and stored at −80°C until usage. The dissected skin samples were then carefully scraped with a curette to selectively remove only the epidermal layer, which was lysed in RIPA buffer containing protease inhibitors. Equal amounts of the lysates were separated on SDS-PAGE and the blots stained with primary antibodies were visualized using IRDye linked secondary antibody in Odyssey SA scanner (LICOR Biosciences, Lincoln, NE) as previously described (Qu et al., 2011 (link)). Antibodies used are specific to Actin (Abcam, Cambridge, MA), GFP (a gift from Dr. Pamela Silver, Harvard Medical School, Boston, MA), Ki67 (BD Pharmingen San Diego, CA), Keratin 10, Keratin 14, phospho-4EBP1 ^T37/46, phospho-S6^S235/236, phospho-Akt^S473, Pten (all from Cell Signaling Technology, Beverly, MA), phospho-Histone H3 (Upstate, Temecula, CA), Fgf1, Fgf2, Fgf7, and Fgf10 (all from Santa Cruz Biotechnology, Santa Cruz, CA). At least three mice of each genotype were analyzed.

Hertzler-Schaefer K., Mathew G., Somani A.K., Tholpady S., Kadakia M.P., Chen Y., Spandau D.F, & Zhang X. (2014). Pten loss induces autocrine FGF signaling to promote skin tumorigenesis. Cell reports, 6(5), 818-826.

+ Open protocol

+ Expand

Characterization of Influenza Virus Proteins

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

Ruxolitinib and PF-4708671 were purchased from Selleck Inc. (Shanghai, China). Gin A and antibodies against phospho-JAK2Y1007/Y1008 (sc-16566-R), β-actin (sc-47778), and GAPDH (sc-166574) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against phospho-tyrosine (P-Y-1000, #8954), STAT3 (#9139), phospho-STAT3Y705 (#9145), JAK2 (#3230), AKT (#4691), phospho-AKTS473 (#4060), S6K1 (#2708), phospho-S6K1T389 (#9234), S6 (#2217), and phospho-S6S235/236 (#4858) were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-nucleoprotein (NP) polyclonal antibody was prepared by immunizing mice with purified recombinant NP protein. An anti-M1 monoclonal antibody was prepared in our lab. An anti-PB2 antibody (#GTX125926) was purchased from GeneTex Inc. (Irvine, CA, USA).

Wang J., Prinz R.A., Liu X, & Xu X. (2020). In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2. Viruses, 12(10), 1141.

+ Open protocol

+ Expand

Curcumin-probe mediated autophagy analysis

Cited 1 time

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

The chemicals used in our experiments were: LysoTracker Red DND-99 (Invitrogen, L7528), Magic RedTM cathepsin B reagent with acridine orange (Immunochemistry Technologies, LLC, 937/6130), CA-074 (APExBIO, A1926), bafilomycin A1 (Baf, Sigma, B1793), acridine orange (AO) (Immunochemistry Technologies, LLC, 6130) and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) (Promega, G5421). The Curcumin-probe (Curcumin-P) was readily synthesized by mono-alkylation of curcumin by propargyl bromide and its structure was verified by H-NMR, C-NMR and high resolution mass spectrometry [40 (link)].
The antibodies used in our experimentsincluded: microtubule-associated protein 1 light chain 3 (LC3) (Sigma, L7543), p62 (Sigma, P0067), Tsc2 (Cell Signaling Technology, 4308), phospho-S6(S235/236) (Cell Signaling Technology, 2211), S6 (Cell Signaling Technology, 2317), β-actin (Sigma, A5441), α-tubulin (Sigma, T6199), Lamin AC (Cell Signaling Technology, 2032), EGF receptor (Cell Signaling Technology, 4267), LAMP1 (Cell Signaling Technology, 9091), PARP1 (Cell Signaling Technology, 9542), TFEB (Bethyl Laboratories, A303-673A), FLAG (Sigma, F1804), 14-3-3 (Cell Signaling Technology, 9638), ANTI-FLAG® M2 Affinity Gel (Sigma, A2220).

Zhang J., Wang J., Xu J., Lu Y., Jiang J., Wang L., Shen H.M, & Xia D. (2016). Curcumin targets the TFEB-lysosome pathway for induction of autophagy. Oncotarget, 7(46), 75659-75671.

+ Open protocol

+ Expand

Western Blot Analysis of Phospho-proteins

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

Immunoblotting was performed as described previously.^{31 (link)} In brief, whole-cell lysates were prepared using boiling lysis buffer (1% SDS, 10 mM Tris·HCl, pH 7.4). Equal amounts of proteins were separated using Criterion or mini gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. The following rabbit antibodies for: phospho-S6 (S235/236), phospho-S6 (S240/244), phospho ERK½, phospho-Thr 389 p70S6K, phospho-AKT (S473) and phospho-AKT (T308), phospho-IRS1 (S1101), phospho-IRS1(S636/639), IRS1 and mouse anti-S6 antibody were from Cell Signaling Biotechnology (Danvers, MA, USA). Rabbit anti-actin and mouse monoclonal anti-GAPDH antibodies were from Sigma-Aldrich and Invitrogen (Camarillo, CA, USA), respectively. Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were from Cell Signaling Biotechnology.

Leontieva O.V., Demidenko Z.N, & Blagosklonny M.V. (2014). Rapamycin reverses insulin resistance (IR) in high-glucose medium without causing IR in normoglycemic medium. Cell Death & Disease, 5(5), e1214-.

+ Open protocol

+ Expand

Reagents for P2X7 Receptor Signaling

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

Carbenoxolone (CBX), BAPTA-AM and thapsigargin (TG) were purchased from Tocris Bioscience (Ellisville, MO). APT102, the recombinant form of soluble CD39, was kindly provided by Dr. Ridong Chen at APT Therapeutics Inc. (St. Louis, MO). All other chemicals and cell culture media were from Sigma-Aldrich (St. Louis, MO) and other culture reagents from Life Technologies (Carlsbad, CA), unless otherwise stated.
Rabbit anti-mouse P2X7 antibody (#APR-004) was obtained from Alomone labs (Jerusalem, Israel); beta-actin (AC-15, #ab6276) from Abcam (Cambridge, MA); Flotillin-2 (B6, #sc-28320) from Santa Cruz Biotechnology, Inc. (Dallas, TX); Caveolin-1 (7C8, #NB100-615) from Novus Biologicals (Littleton, CO); phospho-AKT (S473) (#9271), phospho-PRAS40 (T246) (#2997), phospho-S6K (T389) (#9205), phospho-S6 (S235/236) (#2211) from Cell Signaling Technology (Danvers, MA); HRP-conjugated goat anti-mouse (#31438), donkey anti-rabbit (#31458) and mouse anti-goat IgG (#31400) and the SuperSignal West Femto Maximum Sensitivity Substrate reagents (#PI-34096) were from Thermo Scientific (Rockford, IL).

de Andrade Mello P., Bian S., Savio L.E., Zhang H., Zhang J., Junger W., Wink M.R., Lenz G., Buffon A., Wu Y, & Robson S.C. (2017). Hyperthermia and associated changes in membrane fluidity potentiate P2X7 activation to promote tumor cell death. Oncotarget, 8(40), 67254-67268.

+ 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!