The cells were treated with RANKL (100 ng/ml) with or without activin A (100 ng/ml) for 24 hours. Chromatin immunoprecipitation assay was performed with the simple chromatin immunoprecipitation plus enzymatic chromatin IP kit (Cell Signaling, Danvers, MA) according to the manufacturer’s suggestions using antibodies against histone H3 (1:50 Cell Signaling), normal rabbit IgG (1:100 Cell Signaling), p-c-Fos (1:50 Cell Signaling), or p-Smad2 (1:50 Cell signaling). The purified DNA was analyzed by polymerase chain reaction using primers that detect sequences containing the mouse NFATc1 promoter (forward: 5-CCGGGACGCCCATGCAATCTGTTAGTAATT-3, and reverse: 5-GCGGGTGCCCTGAGAAAGCTACTCTCCCTT-3). All primers were synthesized by Integrated DNA Technologies (Coralville, IA).
P c fos
Manufactured by Cell Signaling Technology
Sourced in United States
P-c-Fos is a primary antibody that detects phosphorylated c-Fos, a transcription factor involved in cellular signaling pathways. It can be used for immunohistochemistry and western blotting applications to study the activation of c-Fos in various cellular and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
P c jun, by Cell Signaling Technology (15 mentions)
C jun, by Cell Signaling Technology (13 mentions)
C fos, by Cell Signaling Technology (12 mentions)
P jnk, by Cell Signaling Technology (6 mentions)
β actin, by Cell Signaling Technology (4 mentions)
P erk1 2, by Cell Signaling Technology (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
P p38 mapk, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (2 mentions)
Pgrmc1, by Cell Signaling Technology (2 mentions)
Fra 1, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Nf κb, by Cell Signaling Technology (2 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Recombinant human il 1β, by R&D Systems (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
18 protocols using p c fos
1
Regulation of NFATc1 Promoter Activity
2
Protein Expression Analysis in Cell Lysates
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The cells of each group were lysed in lysis buffer (#P0013B, Beyotime Biotechnology, China). Determine the quality of the harvested protein by using bicinchoninic acid (BCA) kit (#P0012, Beyotime Biotechnology, China). Then, 20 μg of total proteins was separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred on polyvinylidene difluoride (PVDF) membrane (#ISEQ00010, Millipore, USA) using the semi-dry transfer method. The membranes were blocked for 1 h in Tris-buffered saline containing 5% non-fat dried milk at room temperature (RT) and incubated overnight at 4°C with the relevant antibodies: SLIT2 (#47600), GFP (#55494), p-P38 MAPK (#4511), T-P38 MAPK (#8690), p-C-Fos (#5348), T-C-Fos (#2250), and β-actin (#4970) (1:1,000, all from Cell Signaling Technology, USA). Membranes were rinsed and incubated for 1 h with the corresponding peroxidase-conjugated secondary antibodies (#ab205718, #ab205719, Abcam, USA). Chemiluminescent detection was performed using the enhanced chemiluminescence (ECL) kit (#1251473, Thermo Fisher, USA). Bands were analyzed using ImageJ software (version 1.6 NIH) to verify the relative levels of the above markers. Results are representative of three independent experiments.
3
Protein Extraction and Western Blot Analysis
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Skin was collected from the Acetone and TPA treated wild-type and K14-sPLA2-IIA mice and flash-frozen in liquid Nitrogen. The epidermis was scraped and minced in the RIPA buffer containing protease and phosphatase inhibitors cocktail (Roche). The tissue was homogenized for 10 min and frozen at −80 °C until further use. For the preparation of the whole cell lysates of cultured cells, the cells were washed with ice-cold PBS and scraped in the RIPA buffer. Next, the cell lysate was centrifuged at 16,000 ×g for 30 min at 4 °C. Protein concentration was quantified using Bradford reagent (Sigma) as per manufacturer's instruction. Total 40–60 μg of protein was loaded on 10% SDS-polyacrylamide gel and transferred on to nitrocellulose membrane. Membrane was then blocked with blocking buffer (5% nonfat dry milk or 5% BSA, 10 mM Tris, 150 mM NaCl, 0.1% Tween-20) and probed with the following primary antibodies: p-c-Jun (1:1000), c-Jun (1:1000), p-c-Fos (1:1000), c-Fos (1:1000) all from Cell Signalling Technologies, USA, anti-sPLA2-IIA (1:500) from Merck-Millipore and β-actin (1:10000) from Sigma Aldrich.
4
Immunohistochemical Analysis of Rat Ovary
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Paraffin-embedded rat ovary tissues were prepared, followed by deparaffinization and rehydration. After antigen exposed, tissue slides were incubated with 3% H2O2 for 10 min and goat serum for 30 min to block non-specific binding. Primary antibodies: ANP (1:100; Abcam, Cambridge, UK), NPRA (1:100; Santa Cruz Biotechnology), NPRC (1:100; Santa Cruz Biotechnology), PGRMC1 (1:50; Cell Signaling), PGRMC2 (1:50; Cell Signaling), PCNA (1:200; Proteintech), p-c-Fos (1:50; Cell Signaling), c-Fos (1:50; Cell Signaling), p-c-Jun (1:50; Cell Signaling) and c-Jun (1:50; Cell Signaling) were incubated overnight at 4 °C. The second antibody was incubated for 40 min after washing with PBS. The signals were visualized with DAB wherein yellowish-brown stain indicated as a positive result. The negative control was obtained by replacing primary antibody with PBS. Images were taken under an inverted microscope.
5
Evaluation of Alectinib and Brigatinib Synergy
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APG-2449 was provided by Ascentage Pharm (Rockville, MD). Alectinib and brigatinib were purchased from MedChemExpress (Monmouth Junction, NJ). PS341 was originally provided by Millennium Pharmaceuticals (Cambridge, MA). Human recombinant TRAIL was purchased from PeproTech, Inc. (Rocky Hill, NJ). ERK1/2 and S6 antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). DR5, p-ERK1/2, Akt, p-Akt, p70S6K, p-p70S6K, 4EBP1, p-4EBP1, p-S6, Fra-1, p-Fra-1, c-Jun, p-c-Jun, c-Fos, p-c-Fos and FosB antibodies were all purchased from Cell Signaling Technology, Inc (Beverly, MA). DR4 antibody (B-N28) was purchased from Cell Sciences (Canton, MA). Other reagents and antibodies were the same as described previously [7] (link).
6
Benzidine-induced molecular signaling
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Benzidine (4, 4′-diaminobiphenyl; ≥98.0%, RT), methanol as well as DMSO were purchased from Merck (USA). Benzidine was dissolved in DMSO, which was subsequently preserved at −20°C. The final concentration of DMSO administered in cells was under 1‰. Polyclonal antibodies against p-ERK1/2, p-p38, p-JNK, p-ERK5, p-c-Jun, p-c-Fos, and Fra-1 were purchased from Cell Signaling Technology (USA). Polyclonal antibodies against ZO-1, E-cadherin, Snail, N-cadherin, Vimentin, XMD8-92, U0126, as well as GAPDH were commercially obtained from Santa Cruz (USA). Primers for Snail, N-cadherin, Vimentin, ZO-1, E-cadherin as well as GAPDH were synthesized by Invitrogen (USA). Sources of unmentioned materials were described in the following.
7
Pulsed Electric Field Modulates Inflammatory Pathways
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The pulsed electric field (PEF) was purchased from HVP 5 (Elea, DIL, Quakenbrueck, Germany). Lipopolysaccharide (LPS) and sulforaphane were purchased from Sigma Chemical (St. Louis, MO). Dulbecco's modified Eagle medium (DMEM), Fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for cyclooxygenase 2 (COX-2), β-actin, GAPDH, and histone-3 were obtained from Santa Cruz Biotechnology (Dallas, Texas, United States). The antibody of inducible nitric oxide synthase (iNOS) was purchased from Abcam (Cambridge, UK). The primary antibody for α-tubulin, C-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinases (ERK), NF-κB, I-κB, pI-κB, C-Jun, pC-Jun, C-Fos, and pC-Fos were purchased from Cell Signaling (Beverly, MA, USA). Various ELISA kits like interleukin-6 (IL-6), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), and interleukin-β (IL-1β) were acquired from R&D Systems (Minneapolis, MN, USA).
8
Chondrocyte Culture and Signaling Pathway Analysis
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Media and other reagents for cell culture were purchased from HyClone Laboratories (Logan, UT, USA) or from Life Technologies (Carlsbad, CA, USA). Pronase and Collagenase were from Roche Diagnostics (Indianapolis, IN, USA). Recombinant human IL-1β was from R&D Systems (St Paul, MN, USA). Antibodies specific for p-ERK1/2, ERK1/2, p-JNK, JNK, p-P38, P38, p-c-Fos, c-Fos, p-c-Jun, c-Jun, and Iκβα were purchased from Cell Signaling Technology (Beverly, MA). Antibodies specific for β-Actin, MMP-13, IL-6, iNOS, and COL2A1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-COX-2 antibody was from R&D Systems. Baicalein, Baicalin, Wogonin, Scutellarein and Apigenin were purchased from Extrasynthese (GenayCedex, France).
9
Resveratrol-Enriched Rice Modulates Inflammatory Responses
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Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). LPS, NG-mono-methyl-L-arginine (L-NMMA), and trans-resveratrol were purchased from Sigma-Aldrich (St. Louis, MO, USA). A normal rice (NR) (Oryza sativa var. japonica) and resveratrol-enriched rice (RR) were supplied by the Rural Development Administration (RDA) of South Korea as mentioned in our previous paper [28 (link)]. Enzyme-linked immunosorbent assay (ELISA) development kits for tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and IL-1β were purchased from R&D Systems (Minneapolis, MN, USA). Primary and secondary antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), extracellular signal-regulated kinases (ERK), C-Jun N-terminal kinase (JNK), p38, NF-κB, Histone-3, β-Actin, IkB, pIkB, C-Fos, p-C-Fos, C-Jun, p-C-Jun, and tubulin were purchased from Cell Signaling (Beverly, MA, USA). All other chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO).
10
Protein Expression Analysis by Western Blot
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Protein samples were extracted with lysis buffer. 15 μg protein samples from each group were loaded onto the SDS polyacrylamide gel for electrophoresis and transferred to NC membranes (Millipore, Billerica, MA, USA). After blocking in 5% non-fat milk at room temperature for 2 h, primary antibodies: NPRA (1:1000; Santa Cruz Biotechnology), NPRC (1:1000; Santa Cruz Biotechnology), PGRMC1 (1:1000; Cell Signaling), PGRMC2 (1:1000), Bax (1:2000; Proteintech), Bcl-2 (1:2000; Proteintech), Caspase 8 (1:1000; Proteintech), Caspase 9 (1:1000; Proteintech), PCNA (1:2000; Proteintech), EGFR (1:500; Proteintech), p-EGFR (1:500; Cell Signaling), ERK 1/2 (1:500; Beyotime Biotechnology), p-ERK 1/2 (1:500; Beyotime Biotechnology), p-c-Fos (1:500; Cell Signaling), c-Fos (1:500; Cell Signaling), p-c-Jun (1:500; Cell Signaling), c-Jun (1:500; Cell Signaling) and GAPDH (1:2000; Proteintech) were incubated at 4 °C overnight. Next day, secondary antibodies (1:2000; Cell Signaling) were incubated for 45 min at room temperature. ECL detection system was used to visualize the bands. All experiments were repeated at least three separate times.
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