pCMV6-Alox5-GFP (Origene) was transfected into 293T cells with vectors expressing FLAG-tagged MAPKs. The cells were lysed 24 h later and immunoprecipitated with anti-GFP (Origene), followed by Western blot with anti-phospho-Alox5S217 (Cell Signaling Technology, Danvers, MA, USA). The blots were also probed with anti-GFP. Total lysates were used for Western blot by probing with anti-FLAG (Sigma, St. Louis, MO, USA). JMR cells transfected with siRNA targeting specific genes or non-targeting siRNA control were lysed for Western blot by probing with specific antibodies. The following antibodies were used for Western blot: rabbit polyclonal or monoclonal antibodies to Alox5, ERK1, p38 MAPK, p44/42 MAPK (ERK1/2), phosphor-p44/p42 MAPK (p-ERK1/ERK2), RAD51, SAPK/JNK, SESN2 (Cell Signaling Technology), Alox5AP, DHODH, HADHA, MGST1, TIA-1 (Abcam), EndoG (ProSci), OXR1 (Bethyl Laboratory, Montgomery, TX, USA.) and PDP1 (Sigma); and mouse monoclonal antibodies to AIFM1, API5, PNKP (Santa Cruz Biotechnology), caspase-3, caspase-6 caspase-7, caspse-9, phosphor-p38 MAPK, phosphor-SPAK/JNK (Cell Signaling Technology), FLAG (Sigma) and GFP (Origene). The blots were also probed with mouse monoclonal anti-α tubulin (Santa Cruz Biotechnology) to ensure equal loading.
Phosphor p38 mapk
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Phosphor-p38 MAPK is a laboratory reagent used to detect and quantify the phosphorylation of the p38 mitogen-activated protein kinase (MAPK) in cellular samples. The product provides a specific antibody that recognizes the phosphorylated form of the p38 MAPK protein, allowing researchers to measure the activation status of this important signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
P38 mapk, by Cell Signaling Technology (8 mentions)
Sapk jnk, by Cell Signaling Technology (2 mentions)
Myd88, by Cell Signaling Technology (2 mentions)
Phosphor p44 42, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Rad51, by Cell Signaling Technology (1 mentions)
Sesn2, by Cell Signaling Technology (1 mentions)
Tia 1, by Abcam (1 mentions)
Aifm1, by Santa Cruz Biotechnology (1 mentions)
Mouse monoclonal anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti gfp, by OriGene (1 mentions)
Phosphor akt, by Cell Signaling Technology (1 mentions)
N formyl met leu phe, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
13 protocols using phosphor p38 mapk
1
Assessing MAPK-Alox5 Interactions
2
Signaling Pathways in Neutrophil Activation
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Phorbol ester phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLF), C5a and isoluminol were purchased from Sigma-Aldrich (St. Louis, MO, USA). The p38 MAPK inhibitor SB203580 was ordered from Selleck (Houston, TX, USA). Antibodies including Phosphor-Akt, Akt, Phosphor-p38 MAPK, p38 MAPK, MK2, and GAPDH were ordered from Cell Signaling Technology (Danvers, MA, USA). Anti-p47phox antibody was purchased from Santa Cruz Biotechnology and anti-phospho-p47phox (Ser329) antibody was generated by N.J. Compass Biotechnology (Nanjing, China). Other reagents were ordered from Sigma-Aldrich (St. Louis, MO, USA).
3
Western Blot Analysis of Signaling Proteins
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Western blots were performed as previously described [25 (link),26 (link)]. In brief, heart tissue samples were homogenized in ice-cold lysis buffer and protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA). Heart homogenate proteins were then resolved by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. For reprobing, membranes were stripped with Restore Western Blot Stripping Buffer from Thermo Scientific (Rockford, IL, USA). Rabbit polyclonal antibodies against AMPK, phosphor-AMPK (Thr172), ACC, phosphor-ACC (Ser79), p44/42 MAPK, phosphor-p44/42 MAPK (Thr202/Tyr204), p38 MAPK, phosphor-p38 MAPK (Thr180/Tyr182), SAPK/JNK, phosphor-SAPK/JNK (Thr183/Tyr185) and horseradish peroxidase-linked secondary antibody were purchased from Cell Signaling (Danvers, MA, USA).
4
Whey Protein Hydrolysate Bioactivity Analysis
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WP were supplied by China National Research Institute of Food & Fermentation Industries (Beijing, China). The molecular weights of the WP were 140−1,000 Da and accounted for 92% of the total prepared WP and included a 3−6 amino acid sequence, prepared by hydrolysis of papain method. WP consist of 98.3% protein, 0.05% lipid, 4.56% ash content, and 4.21% water.
The TRIZOL Reagent Kit of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), epidermal growth factor (EGF), interferon-γ (IFN-γ), total antioxidant capacity (TAOC), Aminopeptidase N (APN), interleukin 1β (IL-1β), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha(TNF-α), interleukin-6 (IL-6), zonula occluden (ZO-1), junctional adhesion molecule (JAM-A), and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Kiel biological technology Co.(Shanghai, China). Primary antibodies against TLR4, Myd88, p38MAPK, phosphor-p38MAPK, p44/42, and phosphor-p44/42 were purchased from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase conjugated secondary antibodies and β-actin were purchased from Proteintech.
The TRIZOL Reagent Kit of superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GSH-PX), epidermal growth factor (EGF), interferon-γ (IFN-γ), total antioxidant capacity (TAOC), Aminopeptidase N (APN), interleukin 1β (IL-1β), transforming growth factor-β (TGF-β), tumor necrosis factor-alpha(TNF-α), interleukin-6 (IL-6), zonula occluden (ZO-1), junctional adhesion molecule (JAM-A), and intercellular cell adhesion molecule-1 (ICAM-1) were purchased from Kiel biological technology Co.(Shanghai, China). Primary antibodies against TLR4, Myd88, p38MAPK, phosphor-p38MAPK, p44/42, and phosphor-p44/42 were purchased from Cell Signaling (Beverly, MA, USA). Horseradish peroxidase conjugated secondary antibodies and β-actin were purchased from Proteintech.
5
Inflammatory Response Pathway Regulation
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RPMI-1640 medium, fetal bovine serum (FBS) and antibiotics were obtained from Gibco-BRL (Life Technologies, Grand Island, NY, USA). Dulbecco’s phosphate buffered saline (D-PBS), LPS (from Escherichia coli O111:B4), Griess reagent and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), RIPA buffer, protease inhibitors and phosphatase inhibitors were obtained from Sigma Aldrich (St. Louis, MO, USA). TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from eBioscience (San Diego, CA, USA). BCA protein assay kit and ECL chemiluminescence substrate were obtained from Thermo Scientific (Waltham, MA, USA). Rabbit antibodies against mouse iNOS, COX-2 and â-actin and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit antibodies against mouse phosphor-JNK, JNK, phosphor-ERK, ERK, phosphor-p38 MAPK, p38 MAPK were obtained from Cell Signaling (Farmingdale, NY, USA).
6
Comprehensive Western Blot Analysis
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Western blot analysis was performed as previously described [40 (link)]. Primary antibodies were Collagen I (EMD Millipore), Collagen III (Abcam), Fibronectin (PTG, CHI, USA), ICAM-1 (Abcam), MCP-1 (Abcam), CGRP (Biorbyt, UK) and GAPDH (PTG). ERK 1/2 (44/42kD), phospho-ERK 1/2 (44/42kD), P38-MAPK, phosphor-P38-MAPK, TGF-β, Smad2, phosphor-Smad2, Smad3, phospho-Smad3 antibodies were obtained from Cell signaling technology (CST, Danvers, MA, USA). Secondary antibodies were obtained from Santa Cruz Biotechnology. All experiments were done with at least 3 replications.
7
Western Blot Analysis of Prostate Tissue Proteins
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The frozen prostate tissues were homogenized with T-PER Tissue Protein Extraction Reagent (Thermo Scientific) containing a protease inhibitor (Thermo Scientific). Proteins concentrations were quantified by the Bradford procedure and equal amounts of proteins. Samples were loaded at 30 µg per lane, separated on 12% acrylamide gels, and electroblotted onto nitrocellulose membranes (Hybond-ECL; GE Healthcare UK Ltd., Buckinghamshire, UK). The primary antibodies used in this study were caspase 3, caspase 7, cleaved (cl)-caspase 3, cl-caspase 7, Erk 1/2, phospho-Erk 1/2, p38 Mapk, phosphor-p38 Mapk, and Cyclin D1 (Cell Signaling, Boston, MA, USA) and AR and SV-40 T antigen (Santa Cruz Biotechnology). Equal protein loading was ascertained by Western blotting with β-actin antibody (Sigma-Aldrich). The intensity of each band was measured using the Image J software program, ver. 1.46 (National Cancer Institute Bethesda, MD, USA).
8
Antibody Immunoblot Analysis Protocol
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Antibodies were purchased from the following sources: polyclonal anti-TLR4, anti-PKC-pan (phospho-Thr497), total PKC, phosphor-p38 MAPK, MyD88 were from Cell Signaling Technology (Danvers, MA), the secondary antibodies used for the immunoblot analysis were from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA), DMOG and staurosporine were from Sigma (St. Louis, MO), and TAK-242 was from MedChem Express (Monmouth Junction, NJ).
9
Ginsenoside Rb1 Enhances Stem Cell Therapy
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Ginsenoside Rb1(Rb1) purchased as a reference compound (purity > 98%) from the Division of Chinese Materia Medica and Natural Products, National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), Ministry of Public Health, China. EGM-2MV was purchased from Lonza (Switzerland). DiI-ac-LDL was purchased from Molecular Probe (Thermo Scientific, Waltham, MA, USA). Antibodies against Flk-1, CD133 and CD34 were purchased from Bioss (Beijing, China). Trypsin, penicillin, streptomycin, CM-DiI and FBS were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). Fibronetin was purchased from BD Biosciences (San Jose, CA, USA). L-methionine, SST, FITC-UEA, Hcy, Ficoll, H2DCF-DA, DAPI, Evans blue, AMD3100, SU5416 and GAPDH antibody were purchased from Sigma (St Louis, MO, USA). SDF-1 ELISA Kit was purchased from Cusabio (Wuhan, China). Antibodies against p38MAPK, phosphor-p38MAPK, SDF-1, VEGFR2 and tubulin were purchased from Cell Signaling Technology (Boston, MASS, USA). Fogarty catheter was purchased from Edwards Lifesciences (Irvine, CA, USA).
10
Western Blot Analysis of Signaling Proteins
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Cells were lysed with lysis buffer containing protease inhibitor cocktails (Sigma-Aldrich) as described previously [9] (link),[24] (link). Equal amount of proteins were resolved in 12% SDS-PAGE gel and transferred to PVDF membrane (Millipore, Bedford, MA). The membranes were incubated overnight with antibodies against PAR-1, PAR-2, PAR-3, PAR-4, CTGF (Santa Cruz Biotechnology, Santa Cruz, CA), Col IV, KLK1 (Abcam, Cambridge, UK), phosphor-p42/44 MAPK, p42/44 MAPK, phosphor-p38 MAPK and p38 MAPK (Cell Signaling Technology, Beverly, MA) in 5% non-fat milk, and subsequently incubated with peroxidase conjugated secondary antibodies (Dako, Carpinteria, CA). The immunocomplex was visualized with ECL prime chemiluminescence (GE Healthcare, Buckinghamshire, UK) using ChemiDoc XRS+ system (Bio-Rad, Hercules, CA). Quantification of protein bands was performed by the ImageJ program (NIH, Bethesda, MD) and was normalized to actin level.
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