Ps6 s240 s244

Manufactured by Cell Signaling Technology

Sourced in United States

The PS6 S240/S244 is a laboratory equipment product offered by Cell Signaling Technology. It is designed to measure the phosphorylation of specific serine residues on cellular proteins. The core function of this product is to provide researchers with a tool for analyzing cellular signaling pathways and their associated protein modifications.

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5 protocols using ps6 s240 s244

Nuclear Protein Extraction and Immunoblotting

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Cells were lysed in NP-40 buffer (40 mM HEPES, pH 7.4; 400 mM NaCl; 1 mM EDTA, pH 8.0; 1% NP-40 (CA-630, Sigma); 5% glycerol; 10 mM pyrophosphate; 10 mM β-glycerophosphate; 50 mM NaF; 0.5 mM orthovanadate) containing Protease Inhibitor Cocktail (Sigma) and 1 mM DTT. Nuclear isolation was performed with a Nuclear Extract Kit (40010, Active Motif, Carlsbad, CA, USA), with 10 μg/ml ALLN (208719, Millipore, Bedford, MA, USA) treatment 20 min prior to isolation, and ALLN added to the hypotonic and lysis buffers. The nuclear fraction was washed with hypotonic buffer prior to lysis.
Antibodies used for immunoblots recognized SREBP1 (sc-8984, Santa Cruz, Santa Cruz, CA, USA), SREBP2 precursor and processed C-terminus (557037, BD, Franklin Lakes, NJ, USA), SREBP2 mature N-terminus (30682, Abcam, Cambridge, MA, USA), Actin (A5316, Sigma), and from Cell Signaling Technologies (Danvers, MA, USA): ACC1 (3676), FASN (3180), SCD (2438), HA (2367), P-Akt-T308 (9275), P-Akt-S473 (4051), Total-Akt (4691), P-S6K1-T389 (9234), Total-S6K1 (2708), P-S6-S240/S244 (2215), Total-S6 (2217), 4E-BP1 (9644), Ras (3965), P-Erk1/2-T202/Y204 (9106), Total-Erk1/2 (9102), Lamin A/C (2032), Histone H3 (4499).

Ricoult S.J., Yecies J.L., Ben-Sahra I, & Manning B.D. (2015). Oncogenic PI3K and K-Ras stimulate de novo lipid synthesis through mTORC1 and SREBP. Oncogene, 35(10), 1250-1260.

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Western Blot Analysis of mTOR Pathway

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Western blotting was performed as previously described [41 (link)]. In brief, cell lysates were prepared in NP40 buffer and separated using SDS gel electrophoresis. Afterwards, the proteins were transferred onto a nitrocellulose membrane and incubated with specific primary antibodies against mTOR (#2983, Cell Signaling Technology, Danvers, MA, USA), panAKT (#4685, Cell Signaling Technology), pAKT S473 (#4060, Cell Signaling Technology), S6 (#2217, Cell Signaling Technology), pS6 S240/S244 (#5364, Cell Signaling Technology), pGSK3ß S9 (#9336, Cell Signaling Technology) or HSC70 (#sc-7298, Santa Cruz Biotechnology, Dallas, TX, USA). Afterwards, the membrane was incubated with the appropriate secondary antibodies against anti-mouse-IgG (#7076, Cell Signaling Technology) or anti-rabbit-IgG (#7074, Cell Signaling Technology). The protein expression was analyzed using the LAS-3000 or LAS-4000 Imager from Fuji (Raytest, Straubenhardt, Germany). Densitometric quantification was carried out using AIDA Image Analyser Software Version 3 (Elysia-raytest GmbH, Straubenhardt, Germany).

Moustafa M., Dähling K.K., Günther A., Riebandt L., Smit D.J., Riecken K., Schröder C., Zhuang R., Krech T., Kriegs M., Fehse B., Izbicki J.R., Fischer L., Nashan B., Li J, & Jücker M. (2022). Combined Targeting of AKT and mTOR Inhibits Tumor Formation of EpCAM+ and CD90+ Human Hepatocellular Carcinoma Cells in an Orthotopic Mouse Model. Cancers, 14(8), 1882.

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Western Blotting for Protein Expression

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For western blotting, cells were collected and lysed in 1× SDS‐PAGE loading buffer (1% SDS, 11% glycerol, 10% β‐mercaptoethanol, 0.1 M Tris, pH 6.8) for assay. Protein samples were then probed with corresponding primary/secondary antibodies, illuminated with the Clarity Western ECL substrate (Bio‐Rad, Hercules, CA), and visualized for protein bands with Amersham Imager 600 (GE Healthcare, Chicago, IL). Antibody for HER2 (#4290), p‐S6 (S240/S244, #4858), S6 (#2217), p‐Akt (S473, #9271), Akt (#4691), p‐Erk (T202/Y204, #4370), Erk (#4695), and β‐actin (#4970) were purchased from Cell Signaling technology.

Zhang C., Chen Z., Chong X., Chen Y., Wang Z., Yu R., Sun T., Chen X., Shao Y., Zhang X., Gao J, & Shen L. (2020). Clinical implications of plasma ctDNA features and dynamics in gastric cancer treated with HER2‐targeted therapies. Clinical and Translational Medicine, 10(8), e254.

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Immunohistochemical Analysis of GLUT1 and pS6

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Immunohistochemistry was performed using rabbit polyclonal antibodies against GLUT1 diluted 1:100 (Abcam, Tokyo, Japan) and pS6 (S240/S244) diluted 1:400 (Cell Signaling Technology, Danvers, MA, USA). Briefly, after antigen retrieval and inactivation of endogenous peroxidase, tissue sections were blocked with 2.5% horse serum (ImmPRESS Reagent Kit; Vector Laboratories Inc, Burlingame, CA, USA) at room temperature (RT) for 30 minutes and then incubated at 4°C overnight with antibodies against GLUT1 and pS6 diluted in SignalStain Antibody Diluent (Cell Signaling Technology) and 1% bovine serum albumin/PBS, respectively. After washing in PBS, the sections were incubated at RT for 30 minutes with peroxidase‐conjugated horse anti‐rabbit Ig (ImmPRESS Reagent Kit; Vector Laboratories Inc). Peroxidase activity was detected using ImmPACT DAB Peroxidase Substrate (Vector Laboratories Inc).

Hirashita T., Hirashita Y., Iwashita Y., Endo Y., Kiyonaga M., Matsumoto S., Hijiya N., Moriyama M., Murakami K, & Inomata M. (2020). S6 ribosomal protein phosphorylation is associated with malignancy of intraductal papillary mucinous neoplasm of the pancreas. Annals of Gastroenterological Surgery, 4(5), 571-579.

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Western Blot Analysis of Signaling Proteins

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The following antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA) : EGFR (#2232), pEGFR Y1068 (#2234), HER3 (#4754), pHER3 Y1197 (#4561), pBRAF S445 (#2696), MEK (#9126), pMEK S217/221 (#9154), ERK (#9102), pERK T202/Y204 (#9101), PDK1 (#3062), pPDK1 S241 (#3061), AKT (#9272), pAKT S473 (#9271), pAKT T308 (#9275), ribosomal protein S6 (#2317), pS6 S240/S244 (#5364), FAK (#3285), pFAK Y397 (#8556), SFKs (#2108), pSFKs Y416 (#2101), YES (#3201), Integrin Antibody Sampler Kit (#4749), PARP (#9542), E-cadherin (#3195), Vimentin (#3932), horseradish peroxidase (HRP)-conjugated anti-mouse (#7076) and HRP-conjugated anti-rabbit (#7074). The actin antibody (A2066) was purchased from Sigma-Aldrich. The BRAF antibody (sc-55522) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). For immunoblot, cells were harvested, washed in PBS, and lysed in RIPA buffer [50 mM Tris•HCl (pH 8.0), 150 mM sodium chloride, 5 mM magnesium chloride, 1% Triton X-100. 0.5% sodium deoxycholate, 0.1% SDS, 40 mM sodium fluoride, 1 mM sodium orthovanadate, and complete protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA)]. Western Lightning ECL reagent (Perkin-Elmer) as used for signal detection. Phosphorylated bands were quantified using ImageJ software.

Ichihara E., Westover D., Meador C.B., Yan Y., Bauer J.A., Lu P., Ye F., Kulick A., de Stanchina E., McEwen R., Ladanyi M., Cross D., Pao W, & Lovly C.M. (2017). SFK/FAK signaling attenuates osimertinib efficacy in both drug-sensitive and drug-resistant models of EGFR-mutant lung cancer. Cancer research, 77(11), 2990-3000.

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