Rxxs t

Manufactured by Cell Signaling Technology

Sourced in United States

The RXXS*/T* product is a phospho-specific antibody developed by Cell Signaling Technology. It is designed to detect proteins that have been phosphorylated at arginine-X-X-serine/threonine motifs.

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4 protocols using rxxs t

Western Blot Analysis of Protein Signaling

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Tissue was homogenized, or cultured cells washed, and then lysed in complete RIPA buffer as described previously [50 (link)]. Primary antibodies (Ab) used: AR (sc-816), PI3K p110β (sc-376641), β-actin (sc-47778), GAPDH (sc-32233), and α-tubulin (sc-5286) from Santa Cruz Biotechnology (Santa Cruz, CA); AKT1 (#2938), AKT2 (#3063), AKT3 (#8018), AKT3 (#14982), AKT^poS473 (#9018), AKT^poS473/4 (#9271), AKT2^poS474 (#8599), AKT^poT308 (#4056S), phospho-AKT substrate RXXS*/T* (#9614S), PRAS40 (#2691S), PRAS40^poT346 (#2997T), PTEN (#9552), and PI3K p110α (#4249) from Cell Signaling Technologies (Beverly, MA); SMAD4 (ab-40759) from Abcam (Cambridge, MA). Between 15 and 35 μg of total protein per sample was separated by SDS-PAGE for IB.

Miller K., Degan S., Wang Y., Cohen J., Ku S.Y., Goodrich D, & Gelman I. (2023). PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression. Research Square.

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Kinase Phosphorylation Assay of PKD2 and APOA4

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Recombinant human proteins used for the study, PKD2 and APOA4, were both purchased from Abcam. Kinase reactions were carried out in a kinase reaction buffer containing the immune complex, recombinant proteins, and ATP as described in Bedford et al (2011). Then, Western blot was performed and incubated with a primary antibody against the phosphorylated motif (RxxS/T*; Cell Signaling).

Trujillo‐Viera J., El‐Merahbi R., Schmidt V., Karwen T., Loza‐Valdes A., Strohmeyer A., Reuter S., Noh M., Wit M., Hawro I., Mocek S., Fey C., Mayer A.E., Löffler M.C., Wilhelmi I., Metzger M., Ishikawa E., Yamasaki S., Rau M., Geier A., Hankir M., Seyfried F., Klingenspor M, & Sumara G. (2021). Protein Kinase D2 drives chylomicron‐mediated lipid transport in the intestine and promotes obesity. EMBO Molecular Medicine, 13(5), e13548.

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Kinase Assay for PAH Phosphorylation

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Recombinant GST-PAH (vector synthesized by Vectorbuilder) was produced in Escherichia coli (BL21) as GST fusion protein, and purified by affinity chromatography on glutathione–Sepharose columns. Recombinant human proteins, PKD3 and PKA, were both purchased from Enzo biosciences and SignalChem Biotech, respectively. Kinase reactions were performed in reaction buffer (Cell Signaling Technology) in the presence of cold (nonradioactive) ATP (Cell Signaling Technology) for 30 min at 30°C. As indicated in the experiment, 1 μM of CRT0066101 (Tocris) was added to the corresponding condition. Proteins from the kinase reactions were boiled in 5× Laemmli buffer and analyzed by Western blotting. Membrane was incubated with the appropriate primary antibody against the phosphorylated motif (RxxS/T*) (Cell Signaling Technology), PAH (proteintech, PK), and PKD3 (Cell signaling Technology).

Loza-Valdes A., Mayer A.E., Kassouf T., Trujillo-Viera J., Schmitz W., Dziaczkowski F., Leitges M., Schlosser A, & Sumara G. (2021). A phosphoproteomic approach reveals that PKD3 controls PKA-mediated glucose and tyrosine metabolism. Life Science Alliance, 4(8), e202000863.

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Evaluating PI3K/Akt Pathway Inhibition in Leukemia Cells

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The activity of the PI3K/Akt signalling pathway was evaluated by specific phospho-ELISA kits and confirmed by 2-DE analysis combined with WB using an anti-Akt phospho-substrate (RXXS*/T*, Cell Signalling Technology, Danvers, MA, USA), as previously reported [71 (link),72 (link)]. Synchronized cells were treated for 2 h with the PI3K inhibitor LY294002 (10 µM, Selleck Chemicals, Houston, TX, USA) or the dual PI3K/mTOR inhibitor PF04691502 (1 µM, Merck) and apoptosis was evaluated in C91/PL, C91/III, BJAB and Jurkat cells 24 and 48 h after treatment by flow cytometry after Annexin V/PI staining (Annexin-V-FLUOS Staining Kit, Merck). Two-fold dilutions of the two inhibitors were used to analyse the sensitivity of the four cell lines by MTT assay. Specifically, synchronized cells were seeded in 96-well plates (3 × 10⁴/well) and treated with LY294002 (from 2 μM to 0.25 μM) and PF04691502 (from 20 μM to 2.5 μM) for 48 h. IC₅₀ was determined by a nonlinear regression analysis using the GraphPad Prism software (6.07 for Windows, San Diego, CA, USA).

Rigotto G., Montini B., Mattiolo A., Lazzari N., Piano M.A., Remondini D., Marmiroli S., Bertacchini J., Chieco-Bianchi L, & Calabrò M.L. (2021). Mechanisms Involved in the Promoting Activity of Fibroblasts in HTLV-1-Mediated Lymphomagenesis: Insights into the Plasticity of Lymphomatous Cells. International Journal of Molecular Sciences, 22(19), 10562.

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