Anti-phospho-JNK, anti-JNK, anti-phospho-p38, anti-p38, anti-phospho-MKK3, anti-phospho-MKK4, anti-phospho-ATF2, anti-phospho-MK2, anti-phospho-CREB, anti-phospho-cjun, anti-phospho-IκBɑ, anti-phospho-STAT1, anti-phospho-STAT3, anti-IL-1β, anti-cleaved caspase-3, and anti-phospho-ASK1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ASK1 antibody was provided from Dr. Hidenori Ichijo. Anti-F4/80 antibody was obtained from Serotec. Anti-CD4, anti-caspase-1, and anti-Dclk1 antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-TFF2 antibody was kindly provided by Dr. Sachiyo Nomura. Anti-proton pump and anti-Cdx2 antibodies were obtained from Santa Cruz (Santa Cruz, CA, USA). Anti-BrdU antibody was obtained from Dako (Santa Clara, CA, USA). Anti-actin antibody was purchased from Sigma. Anti-cyclin D1(AB3) was acquired from Invitrogen (Waltham, MA, USA).
Anti phospho p38
Manufactured by Cell Signaling Technology
Sourced in United States, China, Canada
Anti-phospho-p38 is a primary antibody that specifically recognizes the phosphorylated form of the p38 mitogen-activated protein kinase (MAPK) enzyme. p38 MAPK is an important signaling molecule involved in cellular responses to various stimuli, including stress, inflammation, and growth factors.
Automatically generated - may contain errors
Lab products found in correlation
Anti p38, by Cell Signaling Technology (147 mentions)
Anti phospho jnk, by Cell Signaling Technology (106 mentions)
Anti jnk, by Cell Signaling Technology (71 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (65 mentions)
Anti phospho erk, by Cell Signaling Technology (65 mentions)
Anti erk, by Cell Signaling Technology (49 mentions)
Anti erk1 2, by Cell Signaling Technology (47 mentions)
Anti phospho akt, by Cell Signaling Technology (42 mentions)
Anti akt, by Cell Signaling Technology (38 mentions)
Anti β actin, by Cell Signaling Technology (20 mentions)
Anti β actin, by Merck Group (19 mentions)
Anti phospho p65, by Cell Signaling Technology (19 mentions)
Anti p65, by Cell Signaling Technology (17 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (16 mentions)
Anti caspase 3, by Cell Signaling Technology (15 mentions)
Anti gapdh, by Cell Signaling Technology (15 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (14 mentions)
Anti bax, by Cell Signaling Technology (13 mentions)
Anti bcl 2, by Cell Signaling Technology (13 mentions)
Anti iκbα, by Cell Signaling Technology (12 mentions)
249 protocols using anti phospho p38
1
Antibody Characterization for Signaling Pathways
2
Western Blot Analysis of Cell Extracts
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Western blot analysis of cell extracts was performed as described previously (Ohnishi et al. 2017 (link)). Briefly, cells were washed with phosphate-buffered saline three times then lysed in RIPA buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate, and 1.0% (w/v) NP-40 substitute]. After incubation on ice for 20 min, debris was removed by centrifuging at 2000 g for 10 min at 4 °C. Supernatant proteins (20 μg) were electrophoresed with sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Nonspecific binding was blocked by incubating membranes with Blocking One–P (Nacalai Tesque, Kyoto, Japan) for 20 min at room temperature. Then membranes were incubated overnight with primary antibodies, including anti-phospho-Smad1/5/8 (1:1000), anti-Smad1 (1:1000), anti-phospho-ERK1/2 (1:1000), and anti-ERK1/2 (1:1000), anti-phospho-p38 (1:1000), anti-p38 (1:1000) [all from Cell Signaling Technology, Danvers, MA, USA], at 4 °C and then with secondary antibodies for 45 min at room temperature. Immunoreactive protein bands were visualized using an ECL-peroxidase detection system (Thermo Fisher Scientific) and LAS-4000 (Fujifilm, Tokyo, Japan).
3
Investigating β-Actin Regulation Pathways
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βBA, obtained from Sigma-Aldrich (St. Louis, MO, USA), was dissolved and prepared with 5, 10, 20, and 30 mM stock solutions in DMSO. The control group was added 0.1% DMSO. TRIzol reagent was obtained from Life Technologies (Carlsbad, CA, USA). A monoclonal anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against phospho-Akt, anti-Akt, anti-phospho-p38, anti-total p38, anti-phospho-IκB, anti-Bruton’s tyrosine kinase, and anti-phospho-PLCγ2 were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). Anti-phospho-Btk antibody was obtained from GeneTex (Irvine, CA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Donkey anti-rabbit and anti-mouse immunoglobulin secondary antibodies were purchased from Enzo Life Sciences (Farmingdale, NY, USA).
4
Immunoblotting Analysis of Cell Lysates
5
LV Protein Expression Analysis
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The LV tissue was lysed in radioimmunoprecipitation assay (RIPA) lysis buffer, and the total protein was extracted and detected with a BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Approximately 30 μg of total protein was separated by electrophoresis on Laemmli sodium dodecyl sulfate (SDS) polyacrylamide gels. After electrophoresis, the samples were transferred to Immobilon-FL PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk and then incubated with anti-IL-22, anti-IL-22R1 (both from Abcam, Cambridge, England), anti-atrial natriuretic peptide (ANP), anti-B- type natriuretic peptide (BNP), anti-β-myosin heavy chain (β-MHC, triple from Santa Cruz, Dallas, TX, USA), anti-STAT3, anti-phospho-STAT3, anti-ERK, anti-phospho-ERK, anti-c-Jun N-terminal kinase (JNK), anti-phospho-JNK, anti-P38, anti-phospho-P38, and anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH, nine above from Cell Signaling Technology, Boston, USA) antibodies at 4°C overnight. The secondary antibodies were incubated at room temperature for 1 hour. The blots were scanned using a two- color infrared imaging system (Odyssey; LI- COR Biosciences, Lincoln, NE, USA).
6
Protein Expression Analysis in Cardiac Tissue
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In brief, the quantities of protein in samples extracted from cardiomyocytes or the peri‐infarct region of mice were determined using a bicinchoninic acid kit. Equivalent amounts of protein were loaded in equal volumes and fractionated by SDS‐PAGE (10–15% polyacrylamide gels). GAPDH was used as the internal control. The antibodies were as follows: anti‐TGFβR3, anti‐phospho‐p38, anti‐phospho‐ERK1/2, anti‐phospho‐JNK1/2, anti‐p38, anti‐ERK1/2, anti‐JNK1/2 (1:1000 dilution; Cell Signaling Technology), and anti‐GAPDH (1:500 dilutions; Research Diagnostics).
7
Western Blot Analysis of Signaling Proteins
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Proteins were resolved by standard 10% SDS–PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked with 4% bovine serum albumin (w/v) in TBST, and protein bands were detected with specific antibodies using chemiluminescence reagents and a G:BOX Chemi XRQ chemiluminescence imager (Syngene).
The following rabbit antibodies were used: anti‐IκBα (1:1,000; Cell Signaling), anti‐phospho‐p38 (1:1,000; Cell Signaling), anti‐phospho‐SAPK/JNK (1:1,000; Cell Signaling) and anti‐phospho‐ERK (1:1,000; Santa Cruz Biotechnology). Immunoreactive bands were visualised by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit immunoglobulins (1:5,000) or goat anti‐mouse immunoglobulins (1:1,000; Bio‐Rad). To ensure that equal amounts of proteins were loaded, blots were re‐probed with α‐tubulin (1:3,000; Sigma‐Aldrich).
To detect multiple proteins, membranes were re‐probed after stripping of previously used antibodies using a pH 2.2 glycine‐HCl/SDS buffer.
The following rabbit antibodies were used: anti‐IκBα (1:1,000; Cell Signaling), anti‐phospho‐p38 (1:1,000; Cell Signaling), anti‐phospho‐SAPK/JNK (1:1,000; Cell Signaling) and anti‐phospho‐ERK (1:1,000; Santa Cruz Biotechnology). Immunoreactive bands were visualised by incubation with horseradish peroxidase‐conjugated goat anti‐rabbit immunoglobulins (1:5,000) or goat anti‐mouse immunoglobulins (1:1,000; Bio‐Rad). To ensure that equal amounts of proteins were loaded, blots were re‐probed with α‐tubulin (1:3,000; Sigma‐Aldrich).
To detect multiple proteins, membranes were re‐probed after stripping of previously used antibodies using a pH 2.2 glycine‐HCl/SDS buffer.
8
Comprehensive Protein Extraction and Analysis
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Cells were washed with ice-cold Phosphate-Buffered Saline (PBS) and then lysed in Triton X-100-containing lysis buffer. The composition of the lysis buffer was as follows: 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 20 mM Sodium β-Glycerophosphate, 10 mM Sodium Pyrophosphate, 0.5% Triton X-100, Roche protease inhibitor cocktail and 0.1% β-Mercaptoethanol. Lysates were precleared by centrifugation before use for Western blotting. Equal amounts of protein were loaded for Western blot analysis. All the following antibodies used were obtained from Cell Signaling Technology: anti-phospho-p38 (Thr180/Tyr182), anti-p38, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-Mcl-1, anti-phospho-Bad (Ser112), anti-Bad, anti-phospho-MEK1/2, anti-MEK1/2, anti-Grb2, anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-STAT3 (Tyr705), anti-STAT3, anti-calnexin, anti-phospho-p70 S6K1 (Thr389), anti-p70S6K1, except for anti-p27 (BD Biosciences), anti-HIF-1α (BD Transduction Laoratories), anti-GAPDH (US Biological), anti-Glut1 (Abcam), anti-α-tubulin (Molecular Probes), anti-β-actin (Sigma), anti-FLAG M2 (Sigma), anti-V5 (Serotec) and anti-HA (Roche).
9
Investigating Intracellular Signaling Pathways
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Recombinant bovine FGF2, human FGF9, human TGFβ1, mouse noggin/Fc chimera, human BMP4, human BMP6, and anti-BMP antibody (MAB3552) were from R&D Systems (Minneapolis, MN). The following antibodies were all purchased from Cell Signaling Technology (Danvers, MA): anti–phospho-p44/42 MAPK E10 mouse monoclonal (#9106), anti–total p44/42 MAPK (#9102), anti–phospho-p38 (#9211), anti–phospho-MEK1/2 (#9154), anti–phospho Raf-1 (#9427), anti–phospho-FRS2-α(Tyr-196) (#3864), and anti–phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465) (#9511). Other antibodies used were as follows: for CP49, rabbit anti-mouse CP49 polyclonal serum (#899 or #900; both generous gifts of Paul FitzGerald, University of California, Davis, CA); for phospho-tyrosine, 4G10 (a kind gift from Brian Druker, Oregon Health and Science University, Portland, OR); for luciferase, #G745A from Promega (Madison, WI); for GFP, JL-8 from Clontech (Mountain View, CA); for phospho-Smad3, ab51451 from Abcam (Cambridge, MA); for total Smad 1/5, ab75273 from Abcam; for total Raf-1, sc-7267 from Santa Cruz Biotechnology (Santa Cruz, CA); for total p38, sc-535 from Santa Cruz; and for total MEK, M17030 from Transduction Labs (Lexington, KT). UO126 (used at 15 μM), PD173074 (100 nM), and dorsomorphin (5 μM) were from Calbiochem (La Jolla, CA). All other reagents, including TPA, were from Sigma-Aldrich (St. Louis, MO).
10
Western Blot Analysis of HA-Treated Cells
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Whole‐cell extracts from HA‐treated HPF and HGF cells were prepared by lysis in RIPA buffer as described.30 Lysates were run on 10% SDS‐PAGE, and transferred to Amersham™ Protran® membrane (Sigma, Basel, Switzerland). Proteins of interest were visualized using anti‐phospho‐Akt, anti‐Akt, anti‐phospho‐Erk1/2, anti‐Erk, anti‐phospho‐p38, anti‐p38 (all from Cell Signaling Technology, Danvers, MA, USA), and anti‐vinculin (Sigma) antibodies followed by horseradish peroxidase‐conjugated secondary antibodies (MP Biomedicals, Santa Ana, CA, USA) for detection with the SuperSignal™ West Dura Substrate (ThermoFisher Scientific, Zug, Switzerland). Phospho‐Akt, phospho‐Erk1/2 or phospho‐p38 protein expression relative to the respective total protein control was quantified by densitometry using ImageQuant (Molecular Dynamics, Groningen, The Netherlands). Data represent means ± SD from three independent experiments performed with three different cell donors.
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