The following antibodies for histology and cell staining were purchased from Santa Cruz Biotechnology: anti-MUC5AC (Santa Cruz, CA, sc-16903, AB_649616), anti-phospho-PI3K p85α (Tyr467: Santa Cruz, CA, sc-293115, AB_10844180), anti-PI3K p85α (Santa Cruz, CA, sc-31970, AB_2268186), anti-phospho-Akt1 (Thr308: Santa Cruz, CA, sc-135650, AB_2224730), anti-Akt1 (Santa Cruz, CA, sc-1618, AB_630849), anti-phospho-NFκB p65 (Ser536: Santa Cruz, CA, sc-33020, AB_2179018), anti-NFκB p65 (Santa Cruz, CA, sc-109, AB_632039), anti-Lyn (Santa Cruz, CA, sc-15, AB_2281450), and anti-β-actin (Santa Cruz, CA, sc-130656, AB_2223228). The following antibodies for histology were purchased from Abcam Biotechnology: anti-BIP (Abcam, ab21685, AB_2119834), anti-CHOP (Abcam, ab11419, AB_298023), anti-histone H3 (Abcam, ab1791, AB_302613), and anti-IL-13 (Abcam, ab133353, AB_11157609). Anti-phospho-Lyn (Tyr416: Cell Signaling Technology, #2101, AB_331697) was purchased from Cell Signaling Technology. Anti-IL13 (R&D Systems, AF-413-NA, and AB_2124173) and IL-13 ELISA reagents (R&D Systems, M1300CB) were purchased from R&D Systems. IL-13 (PeproTech, #200-13), 4-Phenylbutyric acid (4-PBA, Sigma-Aldrich, P21005), PI3K Inhibitor PI-103 (Selleck, S1038) were purchased as indicated. A nonsilencing siRNA control and a Lyn-specific siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti phospho nf κb p65
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-phospho-NF-κB p65 is a laboratory reagent used to detect the phosphorylated form of the NF-κB p65 subunit. NF-κB p65 is a critical transcription factor involved in regulating immune and inflammatory responses. This antibody can be used in various analytical techniques, such as Western blotting, to study the activation and regulation of the NF-κB signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf κb p65, by Santa Cruz Biotechnology (4 mentions)
Anti phospho jnk, by Santa Cruz Biotechnology (2 mentions)
Anti jnk, by Santa Cruz Biotechnology (2 mentions)
Anti histone h3, by Abcam (1 mentions)
Il 13, by Thermo Fisher Scientific (1 mentions)
4 phenylbutyric acid 4 pba, by Merck Group (1 mentions)
Anti akt1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti lyn, by Santa Cruz Biotechnology (1 mentions)
Anti bip, by Abcam (1 mentions)
Anti muc5ac, by Santa Cruz Biotechnology (1 mentions)
Anti pi3k p85α, by Santa Cruz Biotechnology (1 mentions)
Anti chop, by Abcam (1 mentions)
Anti il 13, by Abcam (1 mentions)
Anti phospho lyn, by Cell Signaling Technology (1 mentions)
Anti il 13, by R&D Systems (1 mentions)
Af 413 na, by R&D Systems (1 mentions)
Anti dapk1, by Merck Group (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti phospho nf κb p65
1
Antibody-Based Histology and Cell Staining Protocols
2
Protein Expression Analysis of dHL-60 and Mouse Neutrophils
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Whole extracts from dHL-60 cells and mouse neutrophils were obtained using a protein extraction reagent kit (KeyGen BioTech). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% SDS gel and then transferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% nonfat milk for 1 h. Next, the membranes were probed with anti-DAPK1 (Sigma-Aldrich), anti-NF-κB p65, anti-phospho-NF-κB p65, anti-Bcl-2, anti-Bax (Santa Cruz, California, USA), anti-phospho-DAPK1 (Cell Signaling Technology, Danvers, MA, USA), or GAPDH antibodies (Antgene Biotechnology, Wuhan, China) overnight at 4 °C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000 dilution; Antgene Biotechnology) for 60 min at room temperature. All proteins were detected by SuperSignal ECL. The band intensities were analyzed by ImageJ (version 1.45s; National Institutes of Health, Bethesda, MD, USA).
3
Immunomodulatory Mechanisms in Murine Models
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IMQ was purchased from InvivoGen (SanDiego, CA, USA). 3-MA was purchased from Sigma-Aldrich (St. Louis, MO, USA). For western blot analysis, anti-β-actin, anti-LC3B, anti-Beclin-1 and anti-Atg5-12 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-p38, anti-p38 and anti-phospho-IκBα were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-phospho-NF-κB p65, anti-NF-κB p65 and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The other reagent sources were as follows: diphenyleneiodonium chloride (DPI) (Calbiochem, USA), apocynin (Sigma-Aldrich, USA), and DCFH-DA (Calbiochem, USA). Anti-CD4-FITC, anti-CD8-FITC, anti-CD11b-FITC, anti-FOXP3-Alexa 488 (126406), anti-Ly6C-APC, anti-CD45 (103124), anti-Ly6G-PE/Cy7 (15-5931-82), anti-TNFα-APC (17-7321-82), anti-IFN-γ-APC (17-7311-82), anti-Gr-1-Alexa 488 (108417) and anti-CD25-Alexa 647 (102020) antibodies were purchased from eBioscience (San Diego, CA, USA). For immunostaining, an anti-LC3 antibody was purchased from MBL International Corporation (Nagoya, Japan), and a FITC-conjugated anti-rabbit secondary antibody was purchased from Jackson Immuno Research (West Grove, PA, USA).
4
Hemin and ZnPP-IX Modulate Cellular Signaling
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). Hemin (ferriprotoporphyrin IX, a potent inducer of HO-1 [13 (link)]) and zinc protoporphyrin IX (ZnPP-IX, a specific HO-1 antagonist [14 (link)]) were the products of Sigma (St. Louis, MO, USA) [15 (link)]. The final concentrations of Hemin and ZnPP-IX were both 0.5 mg/ml. The anti-HO-1 and anti-PPARγ antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-phospho-NF-κB p65 and anti-α-smooth muscle actin (α-SMA) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GW9662 and rosiglitazone were the products of Cayman Chemical (St. Louis, MO, USA) which were used as PPARγ-specific antagonists and agonists, respectively.
5
NF-κB Activation in HaCaT Keratinocytes
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HaCaT keratinocytes were seeded on a sterile coverslip (Marienfeld Superior, Lauda-Königshofen, Germany) in 12-well plate at 5 × 104 cells/mL. After the treatment, cultured keratinocytes were fixed with 4% paraformaldehyde for 10 min at room temperature, followed by blocking with 5% BSA in PBS with permeabilization by 0.1% Triton for 2 h at room temperature. After blocking, the culture was incubated with primary antibodies, anti-phospho-NF-κB p65 at 1:100 (Santa Cruz Biotechnology) overnight at 4°C. The samples were followed by secondary antibodies having incubation for 2 h at room temperature under darkness: 647 (donkey anti-rabbit IgG)-conjugated antibody at 1:200 (Abcam Ltd.) was used. The samples were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Samples were then examined by a Leica SP8 Confocal Microscope (Leica Microsystems, Wetzlar, Germany) with a 63x oil immersion objective.
6
Cell Lysis and Western Blotting for Signaling Pathway Analysis
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Cells were lysed with phopho-lysis buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM EDTA and 10% glycerol] supplemented with 1 mM PMSF, 0.5% Triton X-100, protease inhibitor cocktail, pepstatin A (Sigma-Aldrich), phosphatase inhibitor cocktail (Sigma), and activated sodium orthovanadate (Acros Organics). Three rounds of 5 sec. sonications at 50% amplitude with ultrasonic dismembrator 150T (Fisher Scientific) were performed to facilitate cell lysis. After removing cell debris by centrifugation (3000 x g for 5 min.), supernatants were used for western blotting.
Western blotting was performed, as described previously [41 (link)]. Primary antibodies, anti-Phospho-STAT3 (Tyr 705) (Cell signaling #9131), anti-STAT3 (#9132), anti-Phospho-JNK (#9251), anti-JNK (9252), anti-Phospho-NF-κB p65 (#3033), anti-NF-κB p65 (#4764), anti-GRP78 (Santa Cruz, # SC-166490), anti-CHOP (#SC-7351), anti-gapdh (Millipore, #MAB374), and secondary anti-mouse or anti-rabbit antibodies (Cell signaling) were obtained commercially. Immune-reactive bands were detected by the ImmunStar-AP Substrate (Bio-Rad Laboratories).
Western blotting was performed, as described previously [41 (link)]. Primary antibodies, anti-Phospho-STAT3 (Tyr 705) (Cell signaling #9131), anti-STAT3 (#9132), anti-Phospho-JNK (#9251), anti-JNK (9252), anti-Phospho-NF-κB p65 (#3033), anti-NF-κB p65 (#4764), anti-GRP78 (Santa Cruz, # SC-166490), anti-CHOP (#SC-7351), anti-gapdh (Millipore, #MAB374), and secondary anti-mouse or anti-rabbit antibodies (Cell signaling) were obtained commercially. Immune-reactive bands were detected by the ImmunStar-AP Substrate (Bio-Rad Laboratories).
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
Inflammatory Mediator Quantification
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CA (purity 98%) was obtained from Bordas (Sevilla, Spain), and DSS was obtained from MP Biomedicals (Solon, OH, USA). Dimethyl sulfoxide, hydrogen peroxide, lipopolysaccharide (LPS), olive oil, sodium dodecyl sulfate (SDS), and sulfasalazine (SAL) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals used were of reagent grade. All primary antibodies including anti-S100 calcium-binding protein a8 (S100a8), anti-serum amyloid A3 (Saa3), anti-haptoglobin (Hp), anti-IL-1β, anti-tumor necrosis factor-α (TNF-α), anti-IL-6, anti-CCAAT/enhancer-binding protein β (Cebpb), anti-phospho-p65-NF-κB, anti-phospho-inhibitor of κB (IκB), anti-actin, and secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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