Anti p38α

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-p38α is a primary antibody that recognizes the p38α protein. p38α is a member of the p38 mitogen-activated protein kinase (MAPK) family and plays a central role in the cellular response to various stresses and inflammatory stimuli.

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Anti-p38α MAPK

7 protocols using anti p38α

Western Blot Analysis of p38 MAPK Signaling

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Western blots were performed as described previously [43 ]. Rabbit primary antibodies (Cell Signaling Technology (CST), Danvers, MA) were used at the following dilutions: anti-p38α, -p38β, -p38γ, and -p38δ (1:1000), anti-ß-tubulin (1:2000), anti-p38 GAPDH (1:1000); anti-β-Actin (8H10D10), mouse mAb (1:2000). Anti-DLGH1 and anti-phosphor DLGH1 at serine 158 and 431, total DLGH1 (SAP97) are affinity-purified sheep polyclonal antibodies which were from University of Dundee, Scotland (1:1000). HRP-conjugated goat anti-rabbit (Cat#7074) and anti-mouse IgG, HRP-linked Antibody Cat#7076 (CST, 1:2000) were used as secondary antibodies.
Primary antibodies purchased from CST were used at the following dilutions: p38α (#9218, 1:1000), p38β (#2339, 1:1000), p38δ (#2308, 1:1000), p38γ (#2307, 1:1000), p-p38 Thr180Tyr182 (#4511, 1:1000), GAPDH (#2118, 1:5000), CBP (#7389, 1:1000), p300 (#86377, 1:1000), Ezh2 (#5246, 1:1000), TNFα (#3707, 1:1000), and Anti-SAPK3 (p38γ, ab205926) were purchased from abcam and used at 1:1000 dilution. p-p38 Tyr323 antibody (#12322-1) was purchased from Signalway Antibody and used at 1 μg/mL.

Zhang X.H., Hsiang J, & Rosen S.T. (2021). Flavopiridol (Alvocidib), a Cyclin-dependent Kinases (CDKs) Inhibitor, Found Synergy Effects with Niclosamide in Cutaneous T-cell Lymphoma. Journal of clinical haematology, 2(2), 48-61.

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Investigating p38α and c-MYC Interactions

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Co-immunoprecipitation was carried out as previously described [21 (link)]. The primary antibodies were anti-p38α and anti-c-MYC (both from Cell Signaling, Danvers, MA, USA). IgG was used as a negative control.

Lepore Signorile M., Grossi V., Fasano C., Forte G., Disciglio V., Sanese P., De Marco K., La Rocca F., Armentano R., Valentini A.M., Giannelli G, & Simone C. (2022). c-MYC Protein Stability Is Sustained by MAPKs in Colorectal Cancer. Cancers, 14(19), 4840.

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Immunoblotting Analysis of Signaling Proteins

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Equivalent amounts of protein from the samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto membranes of nitrocellulose. The membranes were then incubated with anti-p65, anti-JNK1/2, anti-p38α or anti-p38β (cat no. 3034, 9252, 9218, 2339, respectively; Cell Signaling Technology, Danvers, MA, USA) or β-actin (cat no. A-1978; Sigma-Aldrich, St. Louis, MO, USA). The antibodies were afterwards detected with anti-rabbit immunoglobulin G (IgG)-HRP (cat no. 7074; Cell Signaling Technology) or with anti-mouse IgG-HRP (cat no. p0447; Dako, Glostrup, Denmark) in a standard ECL reaction (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's instructions.

Bertelsen T., Iversen L, & Johansen C. (2021). I-Kappa-B-Zeta Regulates Interleukin-17A/Tumor Necrosis Factor-Alpha Mediated Synergistic Induction of Interleukin-19 and Interleukin-20 in Humane Keratinocytes. Annals of Dermatology, 33(2), 122-130.

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Western Blot Analysis of Signaling Pathways

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Whole-cell lysates were prepared by lysing adherent macrophages directly in Triton lysis buffer containing a cocktail of protease inhibitors (Roche, USA) and 1µM sodium orthovanadate, separated by SDS-PAGE, and transferred to PVDF membranes as described previously [22 (link)]. Primary antibodies used for western blot analysis included anti-phospho-p38, anti-p38α, anti-phospho-ERK and anti-ERK (Cell Signaling Technologies, Danvers, MA). Anti-mouse and anti-rabbit secondary antibodies were conjugated to DyLight680 and DyLight800, respectively (Jackson ImmunoResearch Laboratories, West Grove, PA). Membranes were imaged using fluorescent detection on the Odyssey CLx instrument (Li-Cor Biosciences, USA), and images were processed using the Image Studio program (Li-Cor Biosciences, USA).

McGill M.M., Sabikunnahar B., Fang Q., Teuscher C, & Krementsov D.N. (2020). The sex-specific role of p38 MAP kinase in CNS autoimmunity is regulated by estrogen receptor alpha. Journal of neuroimmunology, 342, 577209.

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Detecting p38α-MKK3 Interaction by Coimmunoprecipitation

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For detecting the interaction of endogenous p38α with MKK3, co-immunoprecipitation assays were conducted. Briefly, DCs cells treated with OVA@DDAB/PLGA Nv and blank group, lysed in 0.5 ml lysis buffer (50 mM Tris at pH 8.0, 150 mM NaCl, 0.25% NP-40, 1 mM DTT and protease inhibitor tablets from Roche), and immunoprecipitated with anti-p38α (Cell Signaling Technology) or control IgG (Millipore). After extensive washing with lysis buffer, the immuno-precipitates were resolved by SDS–PAGE and were analyzed with Western blot using the anti-MKK3 antibody (Cell Signaling Technology).

Han S., Ma W., Jiang D., Sutherlin L., Zhang J., Lu Y., Huo N., Chen Z., Engle J.W., Wang Y., Xu X., Kang L., Cai W, & Wang L. (2021). Intracellular signaling pathway in dendritic cells and antigen transport pathway in vivo mediated by an OVA@DDAB/PLGA nano-vaccine. Journal of Nanobiotechnology, 19, 394.

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Western Blot Analysis of p38 MAPK Signaling

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Immunoblotting: Antibody Validation Protocol

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The protocol is described elsewhere [27 (link)]. The primary antibodies used were the following: anti-KSRP (Cell Signaling Technology), anti-HuR (Santa Cruz Biotechnology), anti-c-Fos (Santa Cruz Biotechnology), anti-actin (Santa Cruz Biotechnology), anti-β-tubulin (Santa Cruz Biotechnology), anti-α-tubulin (Cell Signaling Technology), anti-haemgglutinin (HA) (Santa Cruz Biotechnology), anti-GFP (Santa Cruz Biotechnology) anti-FLAG (Sigma–Aldrich) and anti-phosphoserine (Millipore), anti-ERK2 (Santa Cruz Biotechnology), anti-ERK5 (Cell Signaling Technology), anti-p38α (Cell Signaling Technology), anti-p38β (Santa Cruz Biotechnology) and anti-p38γ (Santa Cruz Biotechnology) and anti-p38δ (Santa Cruz Biotechnology).

Degese M.S., Tanos T., Naipauer J., Gingerich T., Chiappe D., Echeverria P., LaMarre J., Gutkind J.S, & Coso O.A. (2015). An interplay between the p38 MAPK pathway and AUBPs regulates c-fos mRNA stability during mitogenic stimulation. The Biochemical journal, 467(1), 77-90.

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