T p38 mapk

Manufactured by Cell Signaling Technology

Sourced in United States

T-P38 MAPK is a laboratory product that detects and quantifies phosphorylated p38 MAPK, a key signaling protein involved in cellular stress response pathways. It is used in research applications to study the activation of p38 MAPK under various experimental conditions.

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P38 MAPK (429 citations) P38 (T-P38), (2 citations)

5 protocols using t p38 mapk

Western Blot Analysis of Stress Signaling

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Proteins were extracted with cell lysis buffer containing 1% Triton X-100, sonicated using QSonica-Q800R2, quantified using bicinchoninic acid assay (Sigma) and separated on NuPAGE™ 4-12% Bis-Tris precast gels (Thermo Fisher). Immunoblot analyses were conducted according to standard protocol with the following antisera: caspase-3 (#9662), PARP (#9542), β-actin (#3770), IRE1α (#3294), p-eIF2α (#3597), t-eIF2α (#2103), p-P38 MAPK (#4511), t-P38 MAPK (#8690), p-JNK1/2 (#4668), t-JNK1/2 (#9252), p-ERK1/2 (#9101), t-ERK1/2 (#9102), CHOP (#5554), Bim (#2933), Bcl-2 (#4223), Bax (#5023), p-Akt (#4060), t-Akt (#9272), GAPDH (#5174), cyclin b1 (#12231), p-CDK1 substrates (#9477) and FLAG (#14793) from Cell Signaling Technology; p62 (#ab56416) and Mcl-1 (#ab31948) antibodies were purchased from Abcam, while LC3 antibody (#NB100-2220) was obtained from Novus Biologicals. Images were captured using a ChemiDoc™ Touch Imaging System and processed using ImageLab™ Software.

Trapika I.G., Liu X.T., Chung L.H., Lai F., Xie C., Zhao Y., Cui S., Chen J., Tran C., Wang Q., Zhang S., Don A.S., Li G.Q., Hanrahan J.R, & Qi Y. (2021). Ceramide Regulates Anti-Tumor Mechanisms of Erianin in Androgen-Sensitive and Castration-Resistant Prostate Cancers. Frontiers in Oncology, 11, 738078.

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Protein Expression Analysis in Cell Lysates

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The cells of each group were lysed in lysis buffer (#P0013B, Beyotime Biotechnology, China). Determine the quality of the harvested protein by using bicinchoninic acid (BCA) kit (#P0012, Beyotime Biotechnology, China). Then, 20 μg of total proteins was separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred on polyvinylidene difluoride (PVDF) membrane (#ISEQ00010, Millipore, USA) using the semi-dry transfer method. The membranes were blocked for 1 h in Tris-buffered saline containing 5% non-fat dried milk at room temperature (RT) and incubated overnight at 4°C with the relevant antibodies: SLIT2 (#47600), GFP (#55494), p-P38 MAPK (#4511), T-P38 MAPK (#8690), p-C-Fos (#5348), T-C-Fos (#2250), and β-actin (#4970) (1:1,000, all from Cell Signaling Technology, USA). Membranes were rinsed and incubated for 1 h with the corresponding peroxidase-conjugated secondary antibodies (#ab205718, #ab205719, Abcam, USA). Chemiluminescent detection was performed using the enhanced chemiluminescence (ECL) kit (#1251473, Thermo Fisher, USA). Bands were analyzed using ImageJ software (version 1.6 NIH) to verify the relative levels of the above markers. Results are representative of three independent experiments.

Li P., Lin Z., Liu Q., Chen S., Gao X., Guo W., Gong F., Wei J, & Lin H. (2021). Enhancer RNA SLIT2 Inhibits Bone Metastasis of Breast Cancer Through Regulating P38 MAPK/c-Fos Signaling Pathway. Frontiers in Oncology, 11, 743840.

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Western Blotting Protein Analysis

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Western analysis was performed as described previously (20 (link)). Cell lysates were prepared using complete lysis buffer (EMD Millipore, San Diego, CA) with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Indianapolis, IN). Protein quantification was performed using DC protein assay from Bio-Rad (Hercules, and CA). Western blot analysis was performed as described previously (21 (link),22 (link)). Antibodies used include stromelysin1 (cat. No. 14351-S) dilution 1:1,000 in milk, vascular endothelial cadherin (VE-cadherins; cat No. 2158) dilution 1:1,000 in BSA, P-P38 MAPK (cat No. 9112-S) dilution 1:1,000 in BSA, T-P38MAPK (cat No. 9212-S) dilution 1:1,000 in BSA, P-SRC Tyr-416 (cat No. 6943-S) dilution 1:1,000 in BSA and T-SRC (cat No. 2109-S) dilution 1:1,000 in BSA all from Cell Signaling Technology (Danvers, MA). β-actin (dilution in milk, primary antibodies 1:10,000 and secondary antibodies 1:20,000) from Sigma (St. Louis, MO) and Claudin-5 antibodies (cat No. ab15106) 1:1,000 and secondary antibodies 1:5,000 dilution in milk from Abcam (Cambridge, MA). Band densitometry was done using NIH Image J software.

Kadry R.W., Adil M.S., Newsome A.S, & Somanath P.R. (2021). Cisatracurium attenuates LPS-induced modulation of MMP3 and junctional protein expression in human microvascular endothelial cells. Bioscience trends, 15(1), 50-54.

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Protein Expression Analysis by Western Blot

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Protein extraction was performed with RIPA buffer as described previously [25 ]. Lysates were separated by SDS-PAGE and incubated with antibodies against p-MAPK (Thr202/Tyr204) (Cell Signalling, 4370; 1/1000), t-MAPK 44/42 (Cell Signalling, 4695; 1/1000), DLK-1 antibody (Abcam, ab21682; 1/1000), p-P38MAPK (Cell Signalling, 4511; 1/1000) and t-P38MAPK (Cell Signalling, 8690; 1/1000). Reactive bands were detected by chemiluminescence (Thermo Scientific, Scientific SuperSignal West Femto) and quantified by Image J software. Proteins were normalized to Ponceau S Staining [26 (link)].

Lopez-Tello J., Schofield Z., Kiu R., Dalby M.J., van Sinderen D., Le Gall G., Sferruzzi-Perri A.N, & Hall L.J. (2022). Maternal gut microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice. Cellular and Molecular Life Sciences, 79(7), 386.

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Inflammatory Cytokine Regulation Assay

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Streptomycin (100 ng/mL) and penicillin (100 U/mL) were purchased from Sigma (Gillingham, UK); 96 well plates were provided by NUNC, (Havel, Germany), while GIBCO (Grand island, NY) provided fetal bovine serum (FBS). DMSO, dexamethasone, lipopolysaccharides (LPS—Escherichia coli O111:B4), N-nitro-l-arginine methyl ester (L-NAME), Griess reagent (1% sulfanilamide/0.1% naphthyl ethylenediamine dihydrochloride in 2.5% H₃PO₄), camptothecin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma, IL-6 and TNF-α ELISA Kits were procured from Invitrogen (Massachusetts, US). Synergy Mx microplate reader belonged to BioTek, Winooski, VT, USA. P-IκB, T-IκB, P-p65-NF-κB, T-p65-NF-κB, P-p38-MAPK, and T-p38-MAPK, GAPDH, and anti-rabbit IgG horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Sawhney G., Rasool J.U., Saroch D., Ozturk M., Brombacher F., Ahmad B., Bhagat A., Ali A., Parihar S.P, & Ahmed Z. (2022). Arteannuin-B and (3-Chlorophenyl)-2-Spiroisoxazoline Derivative Exhibit Anti-Inflammatory Effects in LPS-Activated RAW 264.7 Macrophages and BALB/c Mice-Induced Proinflammatory Responses via Downregulation of NF-κB/P38 MAPK Signaling. Molecules, 27(22), 8068.

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