P s6ser240 244

Manufactured by Cell Signaling Technology

Sourced in United States

P-S6Ser240/244 is an antibody that detects phosphorylation of the ribosomal protein S6 at serine residues 240 and 244. This antibody is used to monitor the activity of the mTOR signaling pathway, which regulates protein synthesis and cell growth.

Automatically generated - may contain errors

Lab products found in correlation

17 protocols using p s6ser240 244

Analyzing Protein Signaling Pathways

Cited 1 time

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

Cells were seeded into six-well plates, and media was exchanged the following morning for DMEM containing 0.5% FBS. Cells were harvested at 70% confluency after 72 hours treatment. Western blot analysis of whole cell and tumor lysates was performed, as previously described [21 (link)]. P-AKT^Thr308 (1:1000 dilution), AKT (1:1000 dilution) P-S6^Ser240/244 (1:3000 dilution), S6 (1:1000 dilution), P-JNK ^{Thr183/Tyr185}(1:1000 dilution), JNK (1:1000 dilution), cleaved PARP (1:1000 dilution), BIM (1:1000 dilution), and MCL-1 (1:1000 dilution), were obtained from obtained from Cell Signaling Technologies (Beverly, MA). β-actin (1:5000 dilution) was obtained from Santa Cruz Biotechnology. Secondary antibody (1:10,000 dilution) was obtained from Jackson ImmunoResearch (West Grove, PA).

Roper J., Sinnamon M.J., Coffee E.M., Belmont P., Keung L., Georgeon-Richard L., Wang W.V., Faber A.C., Yun J., Yilmaz O.H., Bronson R.T., Martin E.S., Tsichlis P.N, & Hung K.E. (2014). Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer. Cancer letters, 347(2), 204-211.

+ Open protocol

+ Expand

Western Blot Analysis of Protein Signaling

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

Protein lysates were prepared in 1% CHAPS lysis buffer with protease inhibitor and phosphatase inhibitor, then quantified by BCA assay (Pierce). Samples were run on 10% gels by SDS-polyacrylamide gel electrophoresis, and proteins were transferred by electrophoretic wet transfer to polyvinylidene fluoride membranes. Membranes were blot for p-TBK Ser172 (D52C2), p-AKT Ser473 (D9E) and Thr308 (D25E6), total AKT (C67E7), p-S6K Thr389, p-S6 Ser240/244, β-actin (all Cell Signaling), and HIF-1α (Cayman Chemicals), all diluted to 1:1000 except for HIF-1α (1:500). Incubation with primary antibody overnight was followed by incubation with horseradish peroxidase-linked antibody to rabbit IgG for 45 min. Enhanced chemiluminescence (Perkin Elmer or Amersham) was used to develop the blots. Scans of the original blots are in Supplementary Fig. 7.

Guak H., Al Habyan S., Ma E.H., Aldossary H., Al-Masri M., Won S.Y., Ying T., Fixman E.D., Jones R.G., McCaffrey L.M, & Krawczyk C.M. (2018). Glycolytic metabolism is essential for CCR7 oligomerization and dendritic cell migration. Nature Communications, 9, 2463.

+ Open protocol

+ Expand

Antibody Validation for Protein Analysis

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

The antibodies used for western blot analysis and immunohistochemistry were pAKT
Ser473 (Cell Signaling Technology, 1:1000 dilution), pAKT Thr308 (Cell Signaling
Technology 1:500 dilution), AKT (Cell Signaling Technology, 1:1000 dilution), pS6
Ser240/244 (Cell Signaling Technology, 1:1000 dilution), pERK Thr202/Tyr204 (Cell
Signaling Technology, 1:1000 dilution), ERK (Cell Signaling Technology, 1:1000 dilution),
pMET Tyr1234/1235(Cell Signaling Technology, 1:1000 dilution), MET (Cell Signaling
Technology, 1:1000 dilution), MET (Santa Cruz Biotechnology, 1:200 dilution) and Actin
(Cell Signaling Technology, 1:1000 dilution). All immunohistochemical analyses were
conducted by the MSKCC Molecular Cytology core.

Wanjala J., Taylor B.S., Chapinski C., Hieronymus H., Wongvipat J., Chen Y., Nanjangud G.J., Schultz N., Xie Y., Liu S., Lu W., Yang Q., Sander C., Chen Z., Sawyers C.L, & Carver B.S. (2014). Identifying actionable targets through integrative analyses of GEM model and human prostate cancer genomic profiling. Molecular cancer therapeutics, 14(1), 278-288.

+ Open protocol

+ Expand

Osteoclastogenesis Regulation Mechanism

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

Foetal bovine serum (FBS), α‐minimum essential medium (α‐MEM) and Dulbecco's modified eagle's medium (DMEM) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The JetPRIME® transfection reagent was obtained from Polypus‐transfection (Strasbourg, France). The TRAP staining kit, chloroquine (CQ), antibodies against LC3B and p62, and LY294002 were obtained from Sigma Aldrich (St. Louis, MO, USA). RANKL, OPG and macrophage colony‐stimulating factor (M‐CSF) were obtained from R&D systems (Minneapolis, MN, USA). Antibodies against c‐Fos, Src, PI3K (p85), phosphorylated (p)‐Akt (Ser473), Akt, TAK1, Beclin1 and p‐S6 (Ser240/244) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MMP‐9, and CAⅡ were obtained from Abcam (Beverly, MA, USA). EGFP‐pmCherry‐LC3 plasmid was obtained from HedgehogBio Science and Technology Ltd. (Shanghai, China). Beclin1 siRNA plasmid (sc‐29798) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). BCA protein assay kit and rapamycin (Rap), was obtained from Beyotime (Beijing, China). All related reagents were available in our laboratory.

Tong X., Chen M., Song R., Zhao H., Bian J., Gu J, & Liu Z. (2020). Overexpression of c‐Fos reverses osteoprotegerin‐mediated suppression of osteoclastogenesis by increasing the Beclin1‐induced autophagy. Journal of Cellular and Molecular Medicine, 25(2), 937-945.

+ Open protocol

+ Expand

Immunoblot Analysis of AKT Pathway

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

All antibodies were purchased from Cell Signaling Technology: p-AKT Ser⁴⁷³ (#4060, 1:1000), p-AKT Thr³⁰⁸ (#2965, 1:1000), AKT (#4691, 1:1000), p-PRAS40 Thr²⁴⁶ (#2997, 1:1000), PRAS40 (#2691, 1:1000), PARP (#9542, 1:1000), p-S6 Ser^240/244 (#2215, 1:1000), p-RxxS/T (#9614, 1:1000); HA-tag (#2367, 1:1000); xCT/SLC7A11 (#12691, 1:1000); β-actin (#4970, 1:5000); conformation specific mouse anti-rabbit IgG HRP conjugate (#5127, 1:3000).

Lien E.C., Ghisolfi L., Geck R.C., Asara J.M, & Toker A. (2017). Oncogenic PI3K Promotes Methionine Dependency in Breast Cancer Cells Through the Cystine-Glutamate Antiporter xCT. Science signaling, 10(510), eaao6604.

+ 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

The total protein was extracted from C2C12 myotubes and gastrocnemius muscle using cell lysis buffer for western blotting and IP (Beyotime, China) according to the manufacturer’s protocol. The quantification of total protein was measured with a BCA Protein Assay Kit (Beyotime, China) according to the manufacturer’s protocol.
The primary antibodies to Pan-Akt, P-Akt (Ser473), P-Akt (Thr308), mTOR, P-mTOR (Ser2448), S6, P-S6 (Ser240/244), P70S6K, P-P70S6K (Thr389), 4E-BP1, 4E-BP1 (Ser65) and GAPDH were all purchased from Cell Signaling Technology. Anti-rabbit IgG HRP (ZSGB-Bio, China) was used as a secondary antibody (1:10,000). The blots were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo scientific, USA).

Zou Y., Dong Y., Meng Q., Zhao Y, & Li N. (2018). Incorporation of a skeletal muscle-specific enhancer in the regulatory region of Igf1 upregulates IGF1 expression and induces skeletal muscle hypertrophy. Scientific Reports, 8, 2781.

+ Open protocol

+ Expand

Comprehensive Western Blot Antibody Panel

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

This assay was performed as described previously [14 ]. Primary antibodies used in this study are: ER (6F11) from Abcam, Cambridge, MA, USA; PR (sc-7208) and BCL2 (sc-509) from Santa Cruz Biotechnology, Santa Cruz, CA, USA; P-AKT-Thr308 (#2214) and P-AKT-Ser473 (#2118) from Epitomics, Burlingame, CA, USA; P-PRAS40-Thr246 (#2997), P-GSK3-Ser9 (#9323), P-ERK1/2-Thr202/Tyr204 (#9101), P-S6-Ser240/244 (#2211), P-4EBP1-Thr37/46 (#9459), P-mTOR-Ser2448 (#2971), AKT (#9272), ERK1/2 (#9102), GSK-3β (#9832), PTEN (#9559), β-actin (#4970), and c-PARP (#9541) from Cell Signaling Technology, Danvers, MA, USA. All our shown Western blotting images are from the same gel with the same exposure to allow for a complete comparison between lines and across treatments.

Fu X., Creighton C.J., Biswal N.C., Kumar V., Shea M., Herrera S., Contreras A., Gutierrez C., Wang T., Nanda S., Giuliano M., Morrison G., Nardone A., Karlin K.L., Westbrook T.F., Heiser L.M., Anur P., Spellman P., Guichard S.M., Smith P.D., Davies B.R., Klinowska T., Lee A.V., Mills G.B., Rimawi M.F., Hilsenbeck S.G., Gray J.W., Joshi A., Osborne C.K, & Schiff R. (2014). Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase. Breast Cancer Research : BCR, 16, 430.

+ Open protocol

+ Expand

Evaluating Protein Expression and Modifications

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

Qualitative analysis of protein expression and posttranslational modifications (e.g., lipidation, phosphorylation) was performed as described previously (8 (link)). Lysates (5–30 μg) were separated on bis-acrylamide gels by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to a PVDF membrane and initially stained with ponceau for assessment of total protein loading. Membranes were subsequently probed with antibodies against PDK4 (Abcam #Ab38242), mTOR (Cell Signaling 2983), p-mTOR (Ser2448; Cell Signaling 2971), S6 (Cell Signaling 2317), p-S6^Ser240/244 (Cell Signaling 2215), LC3 (Cell Signaling 12741), p62 (Abnova H00008878-M01), or anti-calsequestrin (Abcam 3516). Following visualization, bands were quantified with the freely available software, ImageJ (NIH). Proteins were normalized to calsequestrin, while phosphoproteins were normalized to their respective total proteins. Importantly, due to the nature of time course studies, in order to minimize the contribution that position on the gel might have on outcomes, samples were randomized on gels; samples were re-ordered post-imaging, only for the sake of illustration of representative images (note, all bands for representative images for an individual experiment were from the same gel).

Brewer R.A., Collins H.E., Berry R.D., Brahma M.K., Tirado B.A., Peliciari-Garcia R.A., Stanley H.L., Wende A.R., Taegtmeyer H., Rajasekaran N.S., Darley-Usmar V., Zhang J., Frank S.J., Chatham J.C, & Young M.E. (2018). Temporal Partitioning of Adaptive Responses of the Murine Heart to Fasting. Life sciences, 197, 30-39.

+ Open protocol

+ Expand

Inhibition of PIM and JAK2 Signaling

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

SET2 or UKE1 cells were treated with either single inhibitor or a combination of PIM inhibitor and JAK2 inhibitor respectively for 4 hours at indicated concentrations. The compounds were then washed off by PBS. The cell pellets were collected and lysed with RIPA buffer (Santa Cruz Biotechnology, Dallas, Texas USA) supplemented with HaltTM phosphatase, protease inhibitor cocktail (Thermo Scientific, Rockford, IL USA,) and phenylmethanesulfonyl fluoride solution (PMSF, Sigma-Aldrich). Lysates were resolved on a 4-12% Bis-Tris gel (Life Technologies, Grand Island, NY, USA) and transferred to nitrocellulose membranes. The membranes were blocked for 1 hour at room temperature with 5% milk, then incubated with the indicated primary antibodies at 1:1000 dilution at 4°C overnight. All primary antibodies were purchased from Cell Signaling (Danvers, MA, USA): Pim1 (2907), Pim2 (4730), Pim3 (4165), c-Myc (5605), pS6-Ser240/244 (2215), S6 (2217), p4EBP1-Thr34/46 (2855), 4EBP1 (9644), pP70S6K-Thr389 (9234), P70S6K (2708), β-actin (5125). After washing, the membranes were incubated with secondary HRP-conjugated anti-rabbit antibody (Santa Cruz) at 1:3,000 for 1 hour at room temperature.

Huang S.M., Wang A., Greco R., Li Z., Sun F., Barberis C., Tabart M., Patel V., Schio L., Hurley R., Chen B., Cheng H., Lengauer C., Pollard J., Watters J., Garcia-Echeverria C., Wiederschain D., Adrian F, & Zhang J. (2014). Combination of PIM and JAK2 inhibitors synergistically suppresses cell proliferation and overcomes drug resistance of myeloproliferative neoplasms. Oncotarget, 5(10), 3362-3374.

+ Open protocol

+ Expand

Comprehensive Protein Analysis Workflow

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

Proteins were extracted using RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM
NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA pH 8.0,
1% NP-40) containing proteinase (Roche) and phosphatase (Roche)
inhibitor cocktails. Samples were resolved using 4–12% SDS-PAGE
gels (Life Technologies) and transferred to PVDF membranes (Millipore).
Membranes were probed overnight on a 4°C shaker with primary antibodies
(1:1,000 dilution unless indicated) recognizing the following proteins: p-AKT
(Ser473) (9271, Cell Signaling), AKT (4691, Cell Signaling), p-S6 (Ser240/244)
(5364, Cell Signaling, 1:20,000), p-4E-BP1 (Thr37/46) (2855, Cell Signaling),
p-AURKA (Thr288) (3079, Cell Signaling), AURKA (4718, Cell Signaling), Cleaved
PARP (Asp214) (9541, Cell Signaling), BCL2 (2870, Cell Signaling), BAX (2772,
Cell Signaling), MYC (ab32072, Abcam), and β-actin (3700, Cell
Signaling).

Donnella H.J., Webber J.T., Levin R.S., Camarda R., Momcilovic O., Bayani N., Shah K.N., Korkola J., Shokat K.M., Goga A., Gordan J.D, & Bandyopadhyay S. (2018). Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer. Nature chemical biology, 14(8), 768-777.

+ 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!