The animals were intraperitoneally injected with insulin (1 U/kg) or saline, and, after 5 min, liver, gastrocnemius muscle, and epididymal adipose tissue were extracted and homogenized in buffer as described in Saad et al. [35] (link). Samples of all tissue extracts were subjected to electrophoresis and western blotting [35] (link), [36] (link), [37] (link). Bands were detected using the chemiluminescence method (West Pico Chemiluminescent Substrate Kit, Thermo Scientific, USA). The antibodies used were anti-phospho-JNK, anti-phospho-IKKα/β, anti-phospho-PERK, anti-phospho-IRE1α, and anti-ATF6α (all obtained from Santa Cruz Technology, Santa Cruz CA, USA) and anti-phospho-Akt and anti-phospho-IRS-1 (Cell Signaling, Boston, MA, USA).
Anti phospho jnk
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-phospho-JNK is a laboratory reagent used for the detection and analysis of phosphorylated c-Jun N-terminal kinase (JNK) in biological samples. It specifically recognizes the phosphorylated form of JNK, which is a key signaling protein involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti jnk, by Santa Cruz Biotechnology (10 mentions)
Anti phospho erk, by Santa Cruz Biotechnology (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Anti p38, by Cell Signaling Technology (4 mentions)
Anti phospho p38, by Cell Signaling Technology (4 mentions)
Anti actin, by Santa Cruz Biotechnology (4 mentions)
Anti phospho p38, by Santa Cruz Biotechnology (3 mentions)
Anti erk1 2, by Cell Signaling Technology (3 mentions)
Anti erk, by Santa Cruz Biotechnology (3 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti phospho erk, by Cell Signaling Technology (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti p38, by Santa Cruz Biotechnology (2 mentions)
Anti lc3, by Novus Biologicals (2 mentions)
Anti beclin 1, by Novus Biologicals (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
22 protocols using anti phospho jnk
1
Insulin Signaling Pathway Analysis
2
Protein Expression Analysis Protocol
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The cells were lysed in RIPA with 1 mM PMSF and protease inhibitors (Sigma) for 15 min on ice. The cytosol and nuclear fraction were isolated according to the manufactory's instruction (Thermo). Similar amounts of protein from each extract were subjected to SDS-PAGE analysis and transferred to polyvinyl difluoride (PVDF) membranes (Millipore). After blocking for 1 h with blocking buffer (5% nonfat milk and 0.1% Tween-20 in PBS), the membranes were incubated with the following primary antibodies at 4°C overnight: anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-JNK, anti-JNK (Santa Cruz), anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt, anti-phospho-IκBα, anti-IκBα, anti-p50/p105, anti-p65, anti-Histone H3 (Cell Signaling Technology), anti-gC1qR (Hycult biotech) and anti-β-actin (Tianjin Sungene Biotech) antibodies. HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz) were used as secondary antibodies. And ECL (Bio-Rad) was used for chemiluminescent detection according to the manufacturer's instructions.
3
Western Blot Analysis of Signaling Pathways
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Cells were harvested in lysis solution containing 50 mM Tris/HCl (pH 7.6), 1 % NP40, 150 mM NaCl, 2 mM EDTA, 100 μM PMSF, a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland), and a phosphatase inhibitor (Sigma-Aldrich). After incubation on ice for 30 min, cellular debris was removed by centrifugation (10 min, 4 °C). Proteins (10 μg) were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The following antibodies were used: anti-β-actin (Sigma-Aldrich), anti-TG2 (Neomarkers, Fremont, CA), anti-E-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA), N-Cadherin (Santa Cruz Biotechnology), anti-phospho-NF-kB p65 (S276) (Cell Signaling, Danvers, MA), anti- NF-kB p65 (Cell Signaling), anti-I-kB (Santa Cruz Biotechnology), anti-phospho-JNK (Santa Cruz Biotechnology), anti-JNK (Santa Cruz Biotechnology), anti-IRAK1 (Cell Signaling), anti-IRAK2 (Cell Signaling), and anti-TRAF6 (Santa Cruz Biotechnology).
4
Murine Dendritic Cell Activation Assay
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Male C57BL/6 mice (4-8-wk-old) were purchased from the Korean Institute of Chemistry Technology (Daejeon, Korea). C57BL/6 OT-I T cell receptor (TCR) transgenic mice, C57BL/6 OT-II TCR transgenic mice, C57BL/6J TLR-2-/- (B6.129- Tlr2tm1Kir/J) mice, and C57BL/10 TLR-4-/- (C57BL/10ScNJ) mice were purchased at 6-8 wk of age from Jackson Laboratory (Bar Harbor, ME). All mice were housed in a specific pathogen-free environment in accordance with the institutional guidelines for animal care. Recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and rmIL-4 were purchased from R&D Systems (Minneapolis, MN). Cytokine enzyme-linked immunosorbent assay (ELISA) kits for murine IL-12p70, tumor necrosis factor-alpha (TNF-α), IL-10, IL-1β, and IL-6 were purchased from R&D Systems. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) were used to detect the expression of CD11c (HL3), CD80 (16-10A1), CD86 (GL1), I-Ab β-chain (AF-120.1), and H-2Kb (AF6-88.5) by flow cytometry. Isotype-matched control mAbs and the biotinylated anti-CD11c (N418) mAb were purchased from R&D Systems. Anti-phospho-ERK, anti-phospho-p38, anti-phospho-JNK, and anti-tubulin Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SB203580, SP600135, and U0126 were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Molecular Profiling of Stem Cell Signaling
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Anti-CD44, anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK1/2, anti-phospho-p38, anti-p38, anti-phospho-SAPK/JNK, and anti-JNK1/2 antibodies were purchased from Cell Signaling Technology (Cambridge, MA, USA). Anti-phospho-JNK, anti-Oct3/4 and anti-β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-SOX2, anti-Notch2, and anti-Snail were purchased from Abcam (Cambridge, MA, USA). Anti-β-catenin was procured from BD Bioscience (Franklin Lakes, NJ, USA). For transfection, non-targeting siRNA and commercial siRNA for SOX2 or Snail or AKT were purchased from Genolution (Genolution Pharmaceuticals, Seoul, Korea). The cells were transfected with each siRNA (50 nM) for 48 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), as described in the manufacturer’s procedure.
6
Protein Expression and Phosphorylation Profiling
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Cells were harvested and lysed with RIPA buffer (Thermo Scientific Inc., Boston, MA, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total protein concentrations were quantified using an RC/DC protein assay reagent (Bio-Rad, Hercules, CA, USA). Protein extracts were separated using 6 and 10% SDS-PAGE and transferred onto PVDF membranes (PALL, Westborough, MA, USA). Membranes were incubated in a blocking solution (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) for 30 min and incubated with anti-VEGF-A, anti-VEGFR-1, anti-VEGFR-2, anti-phospho FAK, anti-phospho ERK1/2, anti-phospho PI3K, anti-phospho AKT, anti-phospho JNK, anti-phospho p38 antibodies (Santa Cruz Biotech. Inc.) at 1:2000 dilution in a blocking solution overnight at 4 °C, and probed with peroxidase conjugated secondary antibodies at 1:5000 dilution. Protein bands were detected using enhanced chemiluminescent Western blotting detection reagent (Thermo Scientific Inc.)
Also, protein extracts were immunoprecipitated using a mouse anti-phospho-Tyr antibody (Santa Cruz Biotech. Inc.) and an ImmunoCruz™ IP/WB Optima kit (Santa Cruz Biotech. Inc.). Immunoprecipitated proteins were subjected to 6% SDS-PAGE and Western blotting using anti-VEGFR-1 and anti-VEGFR-2 antibodies (Santa Cruz Biotech. Inc.)
Also, protein extracts were immunoprecipitated using a mouse anti-phospho-Tyr antibody (Santa Cruz Biotech. Inc.) and an ImmunoCruz™ IP/WB Optima kit (Santa Cruz Biotech. Inc.). Immunoprecipitated proteins were subjected to 6% SDS-PAGE and Western blotting using anti-VEGFR-1 and anti-VEGFR-2 antibodies (Santa Cruz Biotech. Inc.)
7
Bisphenol-A Induced Oxidative Stress
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Otherwise indicated, chemicals and reagents for cell culture were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Bisphenol-A (BPA) and other basic chemicals were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Catalase, superoxide dismutase, and glutathione peroxidase assay kits were from Elabscience (Houston, Texas, USA). Viva cDNA synthesis kit and Luna universal RT-qPCR were from Vivantis (Selangor Darul Ehsan, Malaysia) and New England Biolabs (Ipswich, MA, USA), respectively. The primary antibodies including anti-MHC (Millipore, Billerica, MA, USA), anti-caspase-9, anti-caspase-8, anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-myogenin, anti-phospho-p53, anti-JNK, anti-phospho-JNK, and anti-phospho-P65 NF-κB (Santa Cruz Biotechnology, CA, USA) were also used in this study.
8
Whole-Cell Protein Extraction and Western Blot Analysis
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To prepare whole-cell extracts, cells were scraped from the dishes and suspended in protein extraction buffer (100 mM Tris-Cl (Sigma-Aldrich) pH 7.8, 10 mM NaCl (Sigma-Aldrich), 10% glycerol (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), 50 mM sodium fluoride (Sigma-Aldrich), and 1 mM phenymethylsulfonyl fluoride (Sigma-Aldrich). Equal amounts of protein were separated by electrophoresis on 10% SDS-polyacrylamide gels, followed by electrophoretic transfer to nitrocellulose membranes (EMD Millipore). Membranes were blocked in 5% nonfat dry milk (Amresco LLC) and 0.1% Tween-20 (Amresco LLC) in Tris-buffered saline and probed with the following primary antibodies; the anti-ERK, anti-JNK, anti−p38, anti-phospho-JNK, anti-MEK, anti-FLAG, anti-p53, anti-p21, anti-GSK3β, and anti-ACTIN (Santa Cruz Biotechnology), anti-phospho-ERK, anti-phospho-p38, anti-phospho-GSK3β (Ser9), and anti-phospho-MEK (Cell Signaling). Antibody-antigen complexes were incubated with anti-rabbit or anti-mouse-IgG-peroxidase conjugates (Santa Cruz Biotechnology), followed by detection using an enhanced chemiluminescence (ECL) kit (GE Healthcare Life Sciences).
9
NF1-deficient MPNST Cell Line Culturing
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The NF1-deficient human MPNST cell lines ST88-14 and S462 were cultured in Dulbecco’s Modified Eagle’s Medium (10% fetal bovine serum, 1% penicillin–streptomycin, 1% l -glutamine, and 1% sodium pyruvate) and incubated at 37°C in a humidified atmosphere containing 10% carbon dioxide. No ethical committee approval was required for this set of experiments because the experiments were performed on commercially available cell lines. Dulbecco’s Modified Eagle’s Medium, fetal bovine serum, and tissue culture reagents were obtained from Thermo Fisher Scientific, Waltham, MA, USA. The following antibodies were used: anti-HO-1 (Epitomics, Burlingame, CA, USA), antiphospho-p38 (Cell Signaling Technology, Beverly, MA, USA), antiphospho-ERK-1/2 (Cell Signaling Technology), antiphospho-JNK (Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-p38 (Cell Signaling Technology), anti-ERK-1/2 (Cell Signaling Technology), anti-JNK (Santa Cruz Biotechnology Inc.), and anti-GAPDH (Abcam, Cambridge, MA, USA). All other chemicals and reagents were of analytical grade.
10
Macrophage Activation and Inflammation Regulation
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