P btk

Manufactured by Cell Signaling Technology

Sourced in United States

P-BTK is a monoclonal antibody that detects endogenous levels of BTK protein when phosphorylated at Tyr223. This antibody is a useful tool for studying BTK signaling pathway regulation.

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Lab products found in correlation

P erk, by Cell Signaling Technology (2 mentions) P plcγ2, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Nf κb p65, by Cell Signaling Technology (1 mentions) Protease inhibitor, by Beyotime (1 mentions) Phosphatase inhibitor, by Beyotime (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) P1050, by Beyotime (1 mentions) Polyvinylidene fluoride membranes 0.45 m, by Merck Group (1 mentions) Plcγ2, by Cell Signaling Technology (1 mentions) P nf κb p65, by Cell Signaling Technology (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Ecl solution, by Cytiva (1 mentions) Chemidoc, by Bio-Rad (1 mentions) Cd43 depletion kit, by Miltenyi Biotec (1 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions) Amersham imager, by Cytiva (1 mentions)

Close spelling variants of this product

P-BTK (Y223) (4 citations)

6 protocols using p btk

Western Blot Analysis of Activated B Cells

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Purified CD19 B cells from mouse spleens were stimulated with 10 μg/mL lipopolysaccharide (LPS, Sigma, L2880) for 72 h at 5% CO₂ and 37 °C. The total protein of the activated B cells was then extracted by RIPA lysis buffer (Beyotime, Shanghai, China, P0013B) containing phosphatase inhibitor (Beyotime, P1050) and protease inhibitor (Beyotime, P1011). The prepared protein was separated on 10% SDS-PAGE gels and transferred on polyvinylidene fluoride membranes (0.45 µm; Millipore, Burlington, MA, USA, IPVH00010). Antibodies specific to Btk (Cell Signaling Technology, Danvers, MA, USA, 8547S), pBtk (Cell Signaling Technology, 87141S), PLCγ2 (Cell Signaling Technology, 34264S), pPLCγ2 (Cell Signaling Technology, 3871S), ERK (Cell Signaling Technology, 4695S), pERK (Cell Signaling Technology, 4370S), NF-κB p65 (Cell Signaling Technology, 4764S), pNF-κB p65 (Cell Signaling Technology, 3033S), and GAPDH (Cell Signaling Technology, 8884S) were incubated with the membranes overnight at 4 °C. The membranes were incubated with secondary peroxidase-conjugated antibodies the next day at room temperature for 1 h. Chemiluminescent signals were detected by the ECL method (Beyotime, Shanghai, China, P0018FS). The relative expression of each band was calculated with Fiji software.

Deng G., He J., Huang Q., Li T., Huang Z., Gao S., Xu J., Wang T, & Di J. (2023). Ibrutinib Inhibits BTK Signaling in Tumor-Infiltrated B Cells and Amplifies Antitumor Immunity by PD-1 Checkpoint Blockade for Metastatic Prostate Cancer. Cancers, 15(8), 2356.

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Splenic Naive B Cell Protein Analysis

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Single cell suspensions were generated by pressing spleens through a cell strainer (70 μm) and naïve B cells were purified using a CD43-depletion kit (Miltenyi, 130-049-801). Cells were lysed in 4% SDS containing phosphatase and protease inhibitors (Merck, 4906845001 and 05892970001). Protein concentration was measured by BCA assay (Thermo Fisher, 23225) and concentration was adjusted to 800 μg/ml before the addition of Laemmli buffer. 20 μl per sample were loaded onto 10% polyacrylamide gels and blotted onto a PVDF membrane. Membranes were blocked (5% BSA in TBST) and stained overnight with primary antibody (pp65, Cell Signaling, 3033; pIRAK4, Abnova, MAB2538; pBTK, Cell Signaling, 5082, GAPDH, Cell Signaling, 5174). Membranes were washed and incubated with HRP-coupled secondary antibody for one hour at room temperature. After washing, membranes were incubated with ECL solution (Amersham) and imaged on a ChemiDoc (Bio-Rad). Densitometric analysis was performed using ImageJ.

Flümann R., Rehkämper T., Nieper P., Pfeiffer P., Holzem A., Klein S., Bhatia S., Kochanek M., Kisis I., Pelzer B.W., Ahlert H., Hauer J., da Palma Guerreiro A., Ryan J.A., Reimann M., Riabinska A., Wiederstein J., Krüger M., Deckert M., Altmüller J., Klatt A.R., Frenzel L.P., Pasqualucci L., Béguelin W., Melnick A.M., Sander S., Montesinos-Rongen M., Brunn A., Lohneis P., Büttner R., Kashkar H., Borkhardt A., Letai A., Persigehl T., Peifer M., Schmitt C.A., Reinhardt H.C, & Knittel G. (2020). An Autochthonous Mouse Model of Myd88- and BCL2-Driven Diffuse Large B-cell Lymphoma Reveals Actionable Molecular Vulnerabilities. Blood Cancer Discovery, 2(1), 70-91.

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Quantifying BTK Activation by Autophosphorylation

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Phosphorylation level of BTK at Tyr223 was determined by western blot. Autophosphorylation at Tyr223 within the SH3 domain is necessary for full activation of BTK. Platelet lysates (50 μg) were separated by SDS‐PAGE and transferred to nitrocellulose membranes (Thermo Fisher Scientific). The membranes were then blocked and incubated with anti‐human phosphorylated BTK (p‐BTK) (5082S; Cell Signaling Technology), anti‐human BTK (3533S; Cell Signaling Technology) and anti‐human glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (MAB374; Merck Millipore). Immune complexes were revealed with secondary peroxidase‐conjugated antibodies (Dako A/S). Western blots were analyzed using an Amersham Imager 600 (GE Healthcare). For quantification, densitometry was performed with ImageJ software (National Institutes of Health). The results are shown as the ratio of p‐BTK to total BTK.

Claude L., Martino F., Hermand P., Chahim B., Roger P., de Bourayne M., Garnier Y., Tressieres B., Colin Y., Le Van Kim C., Romana M, & Baccini V. (2022). Platelet caspase‐1 and Bruton tyrosine kinase activation in patients with COVID‐19 is associated with disease severity and reversed in vitro by ibrutinib. Research and Practice in Thrombosis and Haemostasis, 6(8), e12811.

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Protein Lysate Analysis of Cell Signaling

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Total protein lysates were obtained from 1–2 million cells by lysing the samples (1% NP-40 lysis buffer 20 mmol/L Tris-HCl, pH 7.5; 1 mmol/L EDTA; 150 mmol/L NaCl [1% NP-40], containing phosphatase inhibitor cocktails 2 and 3 and protease inhibitors [MilliporeSigma]) for 30–45 minutes at 4°C. Cells in the treatment conditions were either treated with ibrutinib or trametinib (both from Selleck Chemicals) for 1 hour. An equal amount of protein was prepared by calculating the protein concentration using Bradford reagent (Bio-Rad), and 40 μg protein was resolved on 10%–12% SDS-PAGE gel followed by transfer onto a PVDF membrane (MilliporeSigma). Western blot analysis was performed with the following antibodies: ERK (4695, Cell Signaling Technology), p-ERK (4377, Cell Signaling Technology), BTK (8547S, Cell Signaling Technology), p-BTK (87141S, Cell Signaling Technology), CD19 (90176S, Cell Signaling Technology), and β-actin (sc1615 HRP, Santa-Cruz Biotechnology). Ratio analysis was done using Bio-Rad Image Lab software.

Aminov S., Giricz O., Melnekoff D.T., Sica R.A., Polishchuk V., Papazoglu C., Yates B., Wang H.W., Sahu S., Wang Y., Gordon-Mitchell S., Leshchenko V.V., Schinke C., Pradhan K., Aluri S., Sohn M., Barta S.K., Agarwal B., Goldfinger M., Mantzaris I., Shastri A., Matsui W., Steidl U., Brody J.D., Shah N.N., Parekh S, & Verma A. (2024). Immunotherapy-resistant acute lymphoblastic leukemia cells exhibit reduced CD19 and CD22 expression and BTK pathway dependency. The Journal of Clinical Investigation, 134(8), e175199.

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ROR1 and CD19 Immunoprecipitation and Western Blot

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Western blotting was performed using Abs against ROR1, CD19, p-BTK, p-PI3K, p-AKT, p-ERK1/2 (Cell Signaling Technology, Danvers, MA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), according to standard protocols. For immunoprecipitation analysis, cellular lysates were precleared with Protein G Sepharose, incubated with 10 μl ROR1 Ab (Cell Signaling Technology), 10 μl CD19 Ab (Cell Signaling Technology, ), 3.3 μl nonspecific rabbit IgG (Cell Signaling Technology) or 3.3 μl nonspecific mouse IgG (Cell Signaling Technology) overnight at 4°C. Proteins were precipitated by using Protein A Sepharose beads (20 μl) at 4°C for 30 min. The beads were washed five times with 500 μl of 1× lysis buffer (Cell Signaling Technology), followed by Western immunoblotting.

Zhang Q., Wang H.Y., Liu X., Nunez-Cruz S., Jillab M., Melnikov O., Nath K., Glickson J, & Wasik M.A. (2019). ROR1/CD19 receptor complex promotes growth of mantle cell lymphoma cells independently of the B Cell Receptor-BTK signaling pathway. Journal of immunology (Baltimore, Md. : 1950), 203(8), 2043-2048.

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Abivertinib and Apigenin Modulate Signaling Pathways

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Abivertinib (AC0010) was kindly provided by ACEA Pharmaceutical Research (Hangzhou, China), Apigenin was purchased from Selleckchem Company (Houston, USA). Abivertinib and Apigenin were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and sub-packaged in different concentration stored at -20°C. Rabbit polyclonal antibodies to BTK, p-BTK(Tyr223), PLCγ2, p-PLCγ2(Tyr1217), p-IKKα/β(Ser176/180), NF-κB, p-NF-κB, ERK (MAPK), p-ERK (p-MAPK) (Thr202/Tyr204), PI3K, AKT, p-AKT(Ser473), GSK3β,p-GSK3β (Ser9), Bcl-2, Bax, Bcl-xl, Bim, Bad, MCL-1, caspase-3, 7, 8 and PARP were from Cell Signaling Technology (Beverly, MA, USA).

Huang S., Yu M., Shi N., Zhou Y., Li F., Li X., Huang X, & Jin J. (2020). Apigenin and Abivertinib, a novel BTK inhibitor synergize to inhibit diffuse large B-cell lymphoma in vivo and vitro. Journal of Cancer, 11(8), 2123-2132.

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