The HUVECs were lysed with the RIPA solution (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0), to which we added a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA). In each sample, the protein concentration was evaluated using a Bradford assay. Proteins (25 µg) were separated by 4–15% precast polyacrylamide gel (Bio-Rad, Hercules, CA, USA), and then transferred to a 0.2 mm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane, which was then incubated overnight with primary antibodies. Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-ICAM-1 (Cell Signaling), mouse anti-β-actin, mouse anti-α-tubulin, and rabbit-anti-GAPDH (Cell Signaling) were used as the primary antibodies. Anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies were used as the secondary antibodies (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to distinguish the protein bands, using a chemiluminescence substrate (Bio-Rad), that were then quantified using ImageJ software. Each measure was normalized versus β-actin, α-tubulin, or GAPDH.
Mouse anti phospho p38
Manufactured by Cell Signaling Technology
Mouse anti-phospho-p38 is an antibody that specifically binds to the phosphorylated form of the p38 mitogen-activated protein kinase (MAPK). It is used to detect the activation of the p38 MAPK pathway in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by Bio-Rad (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho nf kb, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Mouse anti α tubulin, by Cell Signaling Technology (1 mentions)
Chemiluminescence substrate, by Bio-Rad (1 mentions)
0.2 mm nitrocellulose membrane, by Bio-Rad (1 mentions)
Precast polyacrylamide gel, by Bio-Rad (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Clarity ecl chemiluminescence substrate, by Bio-Rad (1 mentions)
Mouse anti pcna, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti mouse p38, by Cell Signaling Technology (1 mentions)
Ripa cell lysis buffer, by Roche (1 mentions)
Mouse anti iκbα, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Merck Group (1 mentions)
5 protocols using mouse anti phospho p38
1
Evaluating Cellular Signaling Pathways
2
Spinal Cord Protein Expression Analysis
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The dorsal quadrants of L6-S1 spinal cord were harvested and mechanically homogenized and centrifuged. The supernatant was collected and stored at −80℃. The protein concentrations were estimated using the bicinchoninic acid method. Proteins of interest were separated by SDS-PAGE electrophoresis (20 mg of total protein per well) and transferred onto nitrocellulose membranes. The membranes were placed in a blocking solution (Tris-buffered saline with 0.02% Tween and 5% non-fat dry milk powder) for 1 h and incubated overnight with mouse anti-GFAP IgG (1:500, Chemicon), mouse anti-OX42 IgG (1:200, Chemicon), mouse anti-phospho-p38 (1:500, Cell Signaling), rabbit anti- IL-1β IgG (1:300, Endogen) or rabbit anti-P-ser896 NR1 IgG (1:500, Millipore), and mouse anti-β-actin (1:1000, loading control; Sigma). After washing, the membranes were incubated in peroxidase-conjugated secondary antibody (1:10000; Santa Cruz) for 1 h. An enhanced chemiluminescence solution (Pierce, USA) was used to detect the immunocomplexes. Each band was quantified using a computer-assisted imaging analysis system (KONTRON IBAS 2.0).
3
Western Blot Analysis of Signaling Proteins
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RIPA buffer (0.1% SDS, 150 mM NaCl, 1.0% Triton X-100, 10 mM Tris, 5 mM EDTA pH 8.0) with a protease and phosphatase inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) was used to obtain the cell lysates. The Bradford assay was used to evaluate the protein concentration in each sample. Proteins (25 μg) were analyzed by SDS-PAGE, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). A blocking buffer (Bio-Rad, Hercules, CA, USA) was used to block the membrane that was then incubated overnight with primary antibodies.
Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-PCNA (Cell Signaling) and mouse anti-β-actin (Cell Signaling) were used as primary antibodies.
We used secondary horseradish peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to visualize the protein bands by using the Clarity ECL chemiluminescence substrate (Bio-Rad) that were then quantified using ImageJ software. Each measure was normalized with respect to β-actin.
Mouse anti-phospho-p38 (Cell Signaling), rabbit anti-phospho-NF-kB (Cell Signaling), mouse anti-PCNA (Cell Signaling) and mouse anti-β-actin (Cell Signaling) were used as primary antibodies.
We used secondary horseradish peroxidase-conjugated antibodies (anti-mouse or anti-rabbit) (The Jackson laboratory, Bar Harbor, ME, USA). A Uvitec Imager (UVItec, Cambridge, UK) was used to visualize the protein bands by using the Clarity ECL chemiluminescence substrate (Bio-Rad) that were then quantified using ImageJ software. Each measure was normalized with respect to β-actin.
4
Signaling Pathways Activation in Macrophages
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RAW264.7cells were stimulated with rLsa21 and cells were recovered at various time points and treated with RIPA cell lysis buffer supplemented with proteinase and phosphatase inhibitor mixture (Roche Molecular Biochemicals, Indianapolis, IN). The cells were centrifuged at 4 °C, 12,000 × g, for 30 min, and protein concentrations were measured using the BCA protein-quantification assay. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to PVDF membranes (Millipore, Bedford, MA). After blocking with 5% nonfat milk in PBS (pH 7.4, 0.05% (v/v) Tween-20), the membranes were incubated overnight at 4 °C with primary Abs, including rabbit anti-mouse-p38, anti–mouse phospho-p38, anti-mouse-ERK2, anti–mouse phospho-ERK1/2, anti–mouse stress-activated protein kinase/JNK, anti–mouse phospho–stress-activated protein kinase/JNK, anti-mouse IκBα (Cell Signaling Technology, Beverly, MA), according to the manufacturer’s instructions. After washing with PBS, the membranes were incubated for 1–2 h at 37 °C with the appropriate HRP-conjugated anti-rabbit IgG secondary Ab (1:5000, CST, USA). The peroxidase-positive bands were detected using an ECL detection kit.
5
Comprehensive Immunological Assays for In-Depth Analysis
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Gum arabic, activated carbon, 2,7-dichlorofluorescein diacetate (DCFDA), toluidine blue, protease inhibitor cocktail tablets, phosphatase inhibitor cocktail tablets, MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel were purchased from Merck KGaA (Darmstadt, Germany). 25% Aqueous Solution Glutaraldehyde, Paraformaldehyde, Sorensen's Phosphate Buffer, 2% Aqueous Solution Osmium Tetroxide, Ethyl Alcohol, Acetone, Araldite, Dibutyl phthalate (DBP) were purchased Electron Microscopy Sciences (Hatfield, USA). Leukocyte Activation Cocktail with BD GolgiPlug™, FACS antibodies include anti-mouse I-A/I-E-BV510, IgG1-BB700, IgM-BV605, IgE-BV786, IgD-BV711, CD1d-BV421, CD5-PE, CD45-BUV395, CD19-APC, CD45R/B220-BUV496, CD45-APC-Cy7, CD3e-FITC, CD4-V450, CD8-BV510, CD25-BV605, IL-4-PE-Cy7, IFN-γ-PE, FoxP3-AF647, CD103-BUV395, F4/80-BV711, CD80-BV650, CD11b-BV510, Ly6-G-PerCP Cy5.5, PE-Ly6-C, CD45-APC-Cy7, CD11c--AF700 were purchased from BD (Heidelberg, Germany); Another FACS antibody Anti-mouse-IL-17A-BV650 was purchased from eBioscience (Frankfurt am Main, Germany). Anti-mouse HMGB1, Anti-mouse phospho-p65, anti-mouse phospho-p38, anti-mouse GAPDH and anti-rabbit IgG HRP were obtained from Cell signaling Technology (Frankfurt am Main, Germany). IgE and histamine ELISA kits were purchased from Abcam (Berlin, Germany).
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