P38 map kinase

Manufactured by Cell Signaling Technology

Sourced in United States

P38 MAP kinase is a lab equipment product manufactured by Cell Signaling Technology. It is a key enzyme involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway, which plays a crucial role in cellular responses to various environmental stresses and inflammatory signals.

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Close spelling variants of this product

Phospho-p38 MAP kinase (11 citations) Anti‐phospho‐p38 MAP kinase (6 citations) Phospho-specific p38 MAP kinase antibodies (5 citations) Phospho-p38 MAP kinase (Thr180/Tyr182) (5 citations) P38 MAP kinase antibodies (4 citations) Anti-phospho-p38 MAP kinase Thr180/Tyr182 (3 citations) Anti-p38 MAP kinase (2 citations) P38 MAP Kinase assay kit (2 citations) Non-radioactive p38 MAP kinase assay kit (2 citations)

10 protocols using p38 map kinase

Oxidative Stress-Induced Apoptosis Signaling

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β-HB was obtained from Sigma Aldrich (St. Louis, MO, USA). Antibodies directed against caspase-12, phospho-p38 MAP kinase, phospho-SAPK/JNK, p38 MAP kinase, SAPK/JNK, Phospho-ERK1/2, ERK1/2 were obtained from Cell Signaling Technology (Danvers, MA, USA). AIF and ENDOG antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies directed against β-actin were obtained from Sungene Biotech (Nanjing, Jiangsu, China). Antibodies directed against PCNA, caspase-9, caspase-3, bax and bcl-2 were obtained from Boster Biotechnology (Wuhan, Hubei, China). Antibody directed against p53 was obtained from Keygen Biotech (Nanjing, Jiangsu, China). Fura-3/AM and BAPTA/AM were obtained from Dojindo Laboratories (Mashikimachi, Japan). NAC was obtained from beyotime institute of biotechnology (Haimen, Jiangsu, China). All the other reagents, unless otherwise stated, were purchased from Sigma Aldrich (St. Louis, MO, USA).

Tian W., Wei T., Li B., Wang Z., Zhang N, & Xie G. (2014). Pathway of Programmed Cell Death and Oxidative Stress Induced by β-Hydroxybutyrate in Dairy Cow Abomasum Smooth Muscle Cells and in Mouse Gastric Smooth Muscle. PLoS ONE, 9(5), e96775.

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Thymocyte Protein Expression Analysis

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For the sample, the thymus were excised immediately, washed with saline, and weighed. Thymuses were gently homogenized in a glass homogenizer and cells were suspended in Dulbecco’s modified Eagle’s medium and added the chelerythrine chloride and SB203580. For Western blot analysis, cell lysates were prepared in RIPA buffer (50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40), and a protease inhibitor cocktail (Roche). Total protein concentrations were measured using a BCA kit, and proteins were separated in clarified cell extracts by 12% SDS–polyacrylamide electrophoresis. The proteins were the transferred to a nitrocellulose membrane (Millipore) and incubated at 4 °C overnight with primary antibodies for the detection of PON1 (Abcam, Cambridge, MA), p38 MAP kinase (Cell Signalling), p-p38 MAP kinase (Thr180/Tyr182) (Cell Signalling), and Fas (Santa Cruz, USA). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were used for detection of immunoreactive bands and visualized using a chemiluminescent detection system (Santa Cruz, USA).

Bai L., Shi G., Ma Y., Zhang L., Guan F., Zhang X., Xu Y., Chen H, & Zhang L. (2018). Paraoxonase 1 knockout rats have impaired T cell development at the CD4/CD8 double-negative to double-positive transition stage. Scientific Reports, 8, 14457.

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Macrophage Response to r-LdMVK Stimulation

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PBMC-derived macrophages (4 × 10⁶) were seeded and grown for 24 h. Cells were incubated for another 24 h with serum-free RPMI to reduce constitutive signaling. Following starvation, cells were incubated with r-LdMVK (1 ug/ml) from 5 to 90 min. Cells were then washed with PBS containing 2 mM sodium orthovandate (Na₃VO₄) and 5 mM sodium fluoride (NaF) to minimize phosphatase activity. Cell lysis was performed with mammalian lysis buffer (Cell Signaling Technology; #9803) supplemented with phosphatase and protease inhibitors. Protein was quantified by Bicinchonic acid method and probed with antibodies against Phospho-Extracellular signal-regulated kinase (ERK-1/2) (1:1,000 dilution; Cell Signaling Technology, #9101), ERK-1/2 (1:1,000 dilution; Cell Signaling Technology, #9102), Phospho-p38 MAP kinase (1:1,000 dilution; Cell Signaling Technology, #9211), p38 MAP kinase (1:1,000 dilution; Cell Signaling Technology, #9212), Phospho-Cortactin (1:1,000 dilution; Merck, # AB3795), Cortactin (1:1,000 dilution; Cell Signaling Technology, #3502), and house-keeping gene, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1m000 dilution; Santa Cruz Biotechnology, #sc-32233). Each experiment was carried out three times.

Bamra T., Shafi T., Das S., Kumar M., Dikhit M.R., Kumar A., Kumar A., Abhishek K., Pandey K., Sen A, & Das P. (2021). Leishmania donovani Secretory Mevalonate Kinase Regulates Host Immune Response and Facilitates Phagocytosis. Frontiers in Cellular and Infection Microbiology, 11, 641985.

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RNO Modulation of Inflammatory Signaling

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Roflumilast N-oxide (RNO) was provided by Nycomed (Konstanz, Germany). F-12K Nutrient Mixture Kaighn's Modification cell culture medium, antibiotics, glutamine and trypsin-EDTA were purchased from Invitrogen (Eugene, OR, USA). Fetal calf serum (FCS) was from Hyclone (Logan, UT, USA). Lipolysaccharide from E. coli 055:B5, Thiazolyl Blue Tetrazolium Blue (MTT), SB-203580, SP-600125 and U0126 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific antibodies against phospho-(p44/42) ERK1/2, (p44/42) ERK1/2, phospho-p38 MAP kinase, p38 MAP kinase, phospho-SAPK/JNK, SAPK/JNK, were purchased from Cell Signaling Technology (Beverly, MA, USA). Acrylamide, SDS, Tris, HEPES and BSA were purchased from Eurobio (Les Ulis, France). Bradford protein assay and precision plus protein dual color standards were purchased from Bio-Rad (Hercules, CA, USA).

Victoni T., Gleonnec F., Lanzetti M., Tenor H., Valença S., Porto L.C., Lagente V, & Boichot E. (2014). Roflumilast N-Oxide Prevents Cytokine Secretion Induced by Cigarette Smoke Combined with LPS through JAK/STAT and ERK1/2 Inhibition in Airway Epithelial Cells. PLoS ONE, 9(1), e85243.

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Western blotting of target proteins

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Western blotting of the target proteins (n = 5–11 per group) was performed as described elsewhere (Lempiäinen et al. 2013 (link)). Sirtuin 1 (SIRT1) expression was assessed from nuclear proteins. The following antibodies were used: protein kinase B (Akt) (Akt, 1/1000; Cell Signaling, Beverly, MA), pAkt (Phospho-Akt, 1/750; Cell Signaling), AMPK (AMPKalpha, 1/750; Cell Signaling), pAMPK (pAMPKalpha, 1/1000; Cell Signaling), endothelial nitric oxide synthase (eNOS) (NOS3, 1/500; Santa Cruz Biotechnologies, Santa Cruz, CA), eNOS-P (p- NOS3, 1/500; Santa Cruz Biotechnologies), light chain 3B (LC-3B) (LC3B, 1/500; Cell Signaling), p38 (p38 MAP Kinase, 1/1000; Cell Signaling), phospho-p38 (Phospho-p38 MAPK, 1/1000; Cell Signaling), toll like receptor 4 (TLR4) (Anti-TLR4, 1/1000; Abcam, Cambridge, MA), and SIRT1 (Anti-Sir2, 1/1000; Upstate, Millipore, Temecula, CA). Tubulin (Antialpha tubulin, 1/3000; Abcam) and beta-Actin (beta-Actin, 1/3000; Cell Signaling) were used as the loading controls.

Lempiäinen J., Finckenberg P., Mervaala E.E., Storvik M., Kaivola J., Lindstedt K., Levijoki J, & Mervaala E.M. (2014). Dexmedetomidine preconditioning ameliorates kidney ischemia-reperfusion injury. Pharmacology Research & Perspectives, 2(3), e00045.

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Osteoclastogenesis Regulation Protocol

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Recombinant mouse SDF-1α, recombinant soluble mouse M-CSF and RANKL were obtained from PeproTech (Rocky Hill, NJ, USA). AMD3100 8HCl was purchased from Selleck Chemicals (Houston, TX, USA). Rabbit polyclonal antibodies specific for MMP13, MMP-9, cathepsin K, CXCR4, and tartrate-resistant acid phosphatase (TRAP) were purchased from Abcam (Cambridge, MA, USA). Rabbit antibodies against p-Erk1/2, Erk1/2, p-JNK, JNK, p-p38, and p38 MAP kinase were obtained from Cell Signaling Technology (Beverly, MA, USA). The secondary goat or rabbit IgGs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH and RIPA were obtained from Boster (Wuhan, China). Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA, USA).

Dong Y., Liu H., Zhang X., Xu F., Qin L., Cheng P., Huang H., Guo F., Yang Q, & Chen A. (2016). Inhibition of SDF-1α/CXCR4 Signalling in Subchondral Bone Attenuates Post-Traumatic Osteoarthritis. International Journal of Molecular Sciences, 17(6), 943.

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Signaling Pathway Characterization Protocol

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Tramadol hydrochloride, naloxone hydrochloride dihydrate, PGF_2α and hydroxyfasudil (fasudil) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Morphine hydrochloride was obtained from Takeda Pharmaceutical Co., Ltd. (Osaka, Japan). SP600125 and SB203580 were purchased from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). The antibodies against phospho-specific SAPK/JNK, SAPK/JNK, phospho-specific p44/p42 MAP kinase, p44/p42 MAP kinase, phospho-specific p38 MAP kinase, and p38 MAP kinase were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). The antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and myosin phosphatase target subunit (MYPT) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Chalfont, UK). Other materials and chemicals were obtained commercially. PGF_2α was dissolved in ethanol. Tramadol, SP600125 and SB203580 were dissolved in dimethyl sulfoxide. Morphine was dissolved in mast cell medium (150 mM NaCl, 5 mM KCl, 5.5 mM glucose, 0.8 mM MgSO₄, 1 mM CaCl₂ and HEPES, pH 7.4). The maximum concentration of ethanol or dimethyl sulfoxide was 0.1%, which has no effects on the assay for OPG or the detection of the protein level using a Western blot analysis (Kuroyanagi et al., 2014 ).

Kim W., Tanabe K., Kuroyanagi G., Matsushima-Nishiwaki R., Fujita K., Kawabata T., Sakai G., Tachi J., Hioki T., Nakashima D., Yamaguchi S., Otsuka T., Tokuda H., Kozawa O, & Iida H. (2020). Tramadol enhances PGF2α-stimulated osteoprotegerin synthesis in osteoblasts. Heliyon, 6(8), e04779.

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Western Blot Analysis of Stress Signaling

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After transfections and/or treatment with chemicals or not, cells were lysed for the Western Blot assay. Bands were incubated with primary antibodies against SIRT1 (#2493),p16^INK4a (#4824), p21^WAF (#2947), p53 (#9282), MKK6 (#8550),MKK3 (#5674), Phospho-MKK3/MKK6 (#9236), Phospho-p38 MAP Kinase (#9211), p38 MAP Kinase (#9212), Phospho-Hsp27 (#2401), Phospho-MAPKAPK2 (#3007), Phospho-ATF2 (#5112) and actin (#8457) purchased from Cell Signaling Technology (Danvers, MA) and used at the recommended dilution for immunoblotting (1:1000). Raw data presenting full-length blots bands with molecular size markers are shown in Figure S9.

Simoncini S., Chateau A.L., Robert S., Todorova D., Yzydorzick C., Lacroix R., Ligi I., Louis L., Bachelier R., Simeoni U., Magdinier F., Dignat-George F, & Sabatier F. (2017). Biogenesis of Pro-senescent Microparticles by Endothelial Colony Forming Cells from Premature Neonates is driven by SIRT1-Dependent Epigenetic Regulation of MKK6. Scientific Reports, 7, 8277.

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Immunoblotting Analysis of Signaling Pathways

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Proteins (25 μg of soluble lysates) were resolved by SDS–PAGE, transferred to polyvinylidenedifluoride (PVDF) membrane (Millipore), and blotted with antibodies specific to p-ERK (Cell Signaling, Danvers, MA, USA), ERK (Cell Signaling), p-p38 MAP kinase (Cell Signaling), p38 MAP kinase (Cell Signaling), p-JNK (Cell Signaling), JNK (Cell Signaling), IκBα (Cell Signaling), ALOX5 (Abcam, Cambridge, MA, USA), ALOX12 (Abcam), ALOX15-1 (Abcam), ALOX15-2 (Abcam) and actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (31 (link)).

Kwon O., Seo Y, & Park H. (2018). Pinosylvin exacerbates LPS-induced apoptosis via ALOX 15 upregulation in leukocytes. BMB Reports, 51(6), 302-307.

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Western Blot Analysis of Muscle Proteins

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For the analysis of protein expression, muscle samples were separated on a 15% polyacrylamide gel and the proteins transferred to PVDF (Biorad, Ca, USA). The membrane was probed with primary antibodies diluted in Tris buffered saline (TBS) with 0.1% (v/v) Tween-20 and 0.5% (w/v) milk powder. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies before detection of antibody binding by chemiluminescence (Amersham). The membranes were reprobed for anti-b-actin to check protein loading. Band intensity was measured using a BioRad imaging densitometer and quantified using Molecular Analyst software.
Antibodies used were as follows; mouse monoclonal antibodies against Pax7 at 1:1000 myogenin F5D at 1:500 (DSHB) and MyoD 5.8A at 1:1000 (BD Biosciences). Rabbit polyclonal antibodies against Myf5 at 1:1000, MyoD M-318 at 1:1000 and myogenin M-225 (Santa Cruz, TX, USA) at 1:500. p38MAPKinase at 1:1000 and phosphorylated p38MAPKinase (Cell Signalling, USA) at 1:1000. Goat polyclonal antibody against Pax3 (Abcam, MA, USA) at 1:2000. Secondary antibodies were either; anti-mouse IgG (Sigma) at 1:2000, anti-rabbit IgG (Sigma) at 1:2000 or anti-goat IgG (Santa Cruz) at 1:2000.

Lee A.S.J., Yoon N., Gould M, & Zhang M. (2018). Dynamic patterns of mononucleated myogenic cell populations in the developing rat hindlimb. The International journal of developmental biology, 62(4-5).

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