Phosphorylated p38 mapk

Manufactured by Cell Signaling Technology

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Phosphorylated p38 MAPK is a lab equipment product that detects the phosphorylated form of the p38 mitogen-activated protein kinase (MAPK) protein. p38 MAPK is involved in cellular responses to various stimuli, such as stress, cytokines, and growth factors.

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P38 MAPK (429 citations) Phosphorylated p38 (63 citations) Phosphorylated MAPK (5 citations) Phosphorylated p38 MAPK at Thr180/Tyr182 (2 citations) Anti-phosphorylated p38 MAPK (2 citations) Phosphorylated p38MAPK (p-p38 MAPK), (2 citations)

28 protocols using phosphorylated p38 mapk

Quantitative Western Blot Analysis

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Protein samples were prepared using radioimmunoprecipitation assay buffer, and protein concentrations were determined using the BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins (50 μg) were separated on the 12% sodium dodecyl sulfate-polyacrylamide gels, and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Following the blocking with 5% skimmed milk/TBST for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies against Mtdh (1:800; Abcam), p38 MAPK, phosphorylated (p)-p38 MAPK, pro-caspase 3, and cleaved caspase 3 (1:1000; Cell Signaling Technologies, Danvers, MA, USA), β-actin (1:1000; EarthOx LLC, San Francisco, CA, USA), and Bax (1:800; Cell Signaling Technologies). The membranes were probed with the appropriate HRP-conjugated secondary antibodies (1:10 000; EarthOx) for 1 h at room temperature. Protein band intensities were quantified as described previously.^{44 (link)}

Liu W.T., Peng F.F., Li H.Y., Chen X.W., Gong W.Q., Chen W.J., Chen Y.H., Li P.L., Li S.T., Xu Z.Z, & Long H.B. (2016). Metadherin facilitates podocyte apoptosis in diabetic nephropathy. Cell Death & Disease, 7(11), e2477-.

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Western Blot Analysis of p38MAPK

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Amniotic sac and placenta samples were lysed in a RIPA lysis buffer with freshly added protease and phosphatase inhibitors (0.01%). The insoluble material was removed by centrifugation at 10,000 rpm for 20 min at 4°C. The concentration of protein in each tissue lysate was determined by using the BCA protein assay kit (Pierce BCA Protein Assay Kit, Thermo Scientific). The same amount of protein (30 µg) from each sample was loaded onto a 10% SDS-PAGE gel and electrophoresed at 120 V. The resolved proteins were transferred to a PVDF membrane using the iBlot transfer apparatus (Bio-Rad Laboratories). The membranes were blocked in Tris-Buffered Saline (TBS) containing 0.1% Tween 20 (TBS-T) and 5% skim milk for 2h at room temperature. Blots were incubated separately with total p38MAPK (Cell Signaling, Danvers, MA, USA, #9212), phosphorylated (P)-p38MAPK (Cell Signaling, #9211S), or β-actin (Sigma-Aldrich, #A5441) specific primary antibody at 4°C and shaken overnight. Blots were washed three times with TBS-T and incubated with appropriate peroxidase-conjugated IgG secondary antibody for 1h at RT. All blots were developed using chemiluminescence reagents ECL Western Blotting Detection System (Amersham Piscatawya, NJ, USA), in accordance with the manufacturer’s recommendations, followed by autoradiography.

Polettini J., Richardson L.S, & Menon R. (2018). Oxidative Stress Induces Senescence and Sterile Inflammation in Murine Amniotic Cavity. Placenta, 63, 26-31.

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Signaling Protein Analysis in CD34+ Cells

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The following antibodies were used for western blotting: p38 MAPK (product no. 9212), phosphorylated (p-)p38 MAPK (product no. 4511), p44/42 MAPK (product no. 9102), p-p44/42 MAPK (product no. 5726), GSK (product no. 5676), p-GSK (product no. 8566), Akt (product no. 9272), p-Akt (product no. 4060) and β-actin (product no. 4970) were purchased from Cell Signaling Technology, Inc.. PPP was purchased from Santa Cruz Biotechnology, Inc.. PPP was dissolved in DMSO to a concentration of 0.5 mM. In a series of experiments, CD34⁺ cells were incubated with 1 µM PPP in the maintenance medium (StemSpan™ SFEM 09650; StemCell Technologies).

He Q., Zheng Q., Xu F., Shi W., Guo J., Zhang Z., Zhao S., Li X, & Chang C. (2020). IGF-IR promotes clonal cell proliferation in myelodysplastic syndromes via inhibition of the MAPK pathway. Oncology Reports, 44(3), 1094-1104.

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Protein Expression Profiling in HIBECs

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Proteins (40 μg/lane) extracted from each conditioned HIBECs were separated by 15% reducing SDS-PAGE. The proteins were electrotransferred to nitrocellulose membrane (Santa Cruz Biotechnology, Dallas, TX, USA) and incubated with the following antibodies (1:1000 dilutions) overnight at 4 °C: p38 MAPK, phosphorylated (p)-p38 MAPK, ERK1/2, p-ERK1/2, JNK, p-JNK, MKK3/6, p-MKK3/6, p-NF-κB p65, Akt, p-Akt, Src, and p-Src (Cell Signaling Technologies, Danvers, MA, USA), and NF-κB p65, PI3K, p-PI3K and β-actin (Abcam). Anti-human GSTo1 (Santa Cruz Biotechnology) was also incubated as above. To observe correlation between differentially expressed mRNA and protein induction profile, antibodies specific to caspase 8 (CASP8; 1:1000 dilution), B-cell lymphoma 2-related protein A1 (BCL2A1; 1:1000 dilution), Fc fragment of IgM receptor (FCMR; 1:500 dilution), colony-stimulating factor 3 (CSF3; 1:500 dilution) and netrin G2 (NTNG2; 1:1000 dilution) were also proved against each conditioned cell lysate as above. Antibodies were purchased from Abcam. Host-specific peroxidase-conjugated anti-IgG antibodies (1:4000 dilution) were incubated for an additional 2 h. Signals were detected by enhanced chemiluminescence (ECL) (GE Healthcare) after 10 min of exposure. The intensity of each band was quantified by ImageJ software (https://imagej.nih.gov/ij/, accessed on 11 November 2020).

Ahn C.S., Kim J.G., Kang I, & Kong Y. (2021). Omega-Class Glutathione Transferases of Carcinogenic Liver Fluke, Clonorchis sinensis, Modulate Apoptosis and Differentiation of Host Cholangiocytes. Antioxidants, 10(7), 1017.

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Western Blot Analysis of MAPK Signaling

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Total protein lysates from FM explants were collected by tissue homogenization and Western blot analysis was performed as previously described (16 (link)). Membranes were probed with the following primary antibodies from Cell Signaling Technology (Danvers, MA): phosphorylated (p) p38 MAPK (#9211S; 1:10,000 dilution); total (t) p38 MAPK (#9212; 1:30,000 dilution); p-ERK (#9101; 1:1000 dilution); and t-ERK (#4695S; 1:2000 dilution). Chemiluminescence was detected using an Amersham Imager 680 (General Electric, Boston, MA) and semi-quantitative densitometry was performed using the Gel Logic 100 (Eastman Kodak, Rochester, NY) and Carestream software (Carestream Molecular Imaging, CT). Levels of phosphorylated protein were normalized against the total amount of that specific protein.

Tong M., Smith A.H, & Abrahams V.M. (2021). Activated Neutrophils Propagate Fetal Membrane Inflammation and Weakening through ERK and Neutrophil Extracellular Trap-induced TLR-9 Signaling. Journal of immunology (Baltimore, Md. : 1950), 206(5), 1039-1045.

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Propofol Cytotoxicity and Apoptosis Regulation

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Propofol (purity >97%; Lot number: D126608) was obtained from Sigma Aldrich (St. Louis, USA). A Cell Counting Kit-8 (CCK-8) was purchased from Dojindo laboratories (Kumamoto, Japan). A bicinchoninic acid (BCA) Protein Assay Kit was obtained from the Beyotime Institute of Biotechnology (Haimen, China). The Colorimetric CaspACE™ Assay System was purchased from Promega (Madison, WI, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rabbit polyclonal antibodies for Akt, phosphorylated (p)-Akt (Ser473), p38MAPK, phosphorylated (p)-p38MAPK, JNK, phosphorylated (p)-JNK, Bax, Bcl-2, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). Dimethyl sulfoxide (DMSO) was purchased from Biosharp (Hefei, China). All other chemicals and reagents used were commercially available and of standard biochemical quality.

Xie C.L., Pan Y.B., Hu L.Q, & Qian Y.N. (2015). Propofol attenuates hydrogenperoxide-induced apoptosis in human umbilical vein endothelial cells via multiple signaling pathways. Korean Journal of Anesthesiology, 68(5), 488-495.

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Adiponectin-Mediated Cell Signaling Analysis

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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Thermo Scientific, Waltham, MA, USA. Hank's balanced salt solution (HBSS), rat recombinant globular adiponectin (gAd), Z-VAD-FMK, necrostatin-1, dihydroethidium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide kit were purchased from Sigma-Aldrich, St. Louis, MO, USA. Antibodies against cleaved caspase-3, caspase-3, RIP1, RIP3, NF-κB, p38MAPK, phosphorylated-NF-κB, phosphorylated-p38MAPK, Bcl-2, Bax, and GAPDH as well as HRP-conjugated anti-rabbit IgG antibody were obtained from Cell Signaling Technology, Inc., Danvers, MA, USA.

Zhu K., Guo J., Yu X., Wang Q., Yan C., Qiu Q., Tang W., Huang X., Mu H., Dou L., Bian Y., Han Q., Shen T., Li J, & Xiao C. (2021). Polypeptide Globular Adiponectin Ameliorates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Inhibiting Both Apoptosis and Necroptosis. Journal of Immunology Research, 2021, 1815098.

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Western Blot Analysis of Phospho-p38 MAPK

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Cells were lysed with Laemmli buffer and total protein concentration determined. A total 15 μg of total protein from each lysate was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a Polyvinylidene fluoride (PVDF) membrane (Invitrogen). The membranes were probed with the following primary antibodies: α-tubulin (1:2000 Sigma), total p38–MAPK (1:1000 Santa Cruz), phosphorylated p38–MAPK (1:1000 Cell Signaling). Secondary antibodies: HRP-conjugated goat anti-mouse and goat anti-rabbit IgG (H+L) (1:10 000, Jackson Laboratories).

Mogilevsky M., Shimshon O., Kumar S., Mogilevsky A., Keshet E., Yavin E., Heyd F, & Karni R. (2018). Modulation of MKNK2 alternative splicing by splice-switching oligonucleotides as a novel approach for glioblastoma treatment. Nucleic Acids Research, 46(21), 11396-11404.

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Evaluating Oxidative Stress and Apoptosis

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Cell counting kit-8 (CCK-8) and Annexin V-FITC/PI apoptosis detection kits were purchased from Vazyme Biotechnology Company (Nanjing, China). T-BHP, dimethyl sulfoxide (DMSO), and 2′,7′-dichlorodihydrofluorescein diacetate (DCFHDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cisplatin and N-acetylcysteine (NAC) were purchased from Macklin Biochemical Corporation (Shanghai, China). CpG ODN1826 was purchased from InvivoGen (San Diego, CA, USA). The JC-1 kit was purchased from Beyotime (Shanghai, China). RAW264.7 and AML12 (alpha mouse liver 12) cells were received from American Type Culture Collection (Manassas, VA, USA). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and Opti-MEM were purchased from Gibco (Waltham, MA, USA). Antibodies for cleaved-caspase 3, caspase 3, caspase 8, cleaved-caspase 8, caspase 9, cleaved-caspase 9, PARP, cleaved PARP, phosphorylated ERK1/2 (p-ERK1/2), ERK1/2, phosphorylated Akt (p-Akt), phosphorylated p38 MAPK, p38 MAPK, phosphorylated JNK, JNK, anti-rabbit IgG, anti-mouse IgG, and GAPDH were all purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NOX2 antibody (ab80508) was purchased from Abcam (Cambridge, MA, USA). AST and ALT detection kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Qu Y., Yang C., Li X., Luo H., Li S., Niu M., Chen P., Yan Z, & Jiang Y. (2020). CpG-Oligodeoxynucleotides Alleviate Tert-Butyl Hydroperoxide-Induced Macrophage Apoptosis by Regulating Mitochondrial Function and Suppressing ROS Production. Oxidative Medicine and Cellular Longevity, 2020, 1714352.

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SDS-PAGE and Immunoblotting Protocol

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We followed the standard protocol for SDS-PAGE electrophoresis, membrane transferring, antibody incubation, and Chemluminescence development (ECL, Millipore) [33 (link)]. The antibodies used in this study, VDAC, phosphorylated p38 MAPK, and GAPDH were all purchased from Cell Signaling Technology (CA, USA).

He J., Lu Y., Xia H., Liang Y., Wang X., Bao W., Yun S., Ye Y., Zheng C., Liu Z, & Shi S. (2015). Circulating Mitochondrial DAMPs Are Not Effective Inducers of Proteinuria and Kidney Injury in Rodents. PLoS ONE, 10(4), e0124469.

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