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Rabbit anti p65

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Rabbit anti-p65 is a primary antibody that recognizes the p65 subunit of the NF-κB transcription factor. It can be used to detect and quantify the expression of p65 in various biological samples.

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Lab products found in correlation

Rabbit anti p p65, by Cell Signaling Technology (11 mentions) Rabbit anti phospho p65, by Cell Signaling Technology (7 mentions) Bca protein assay kit, by Thermo Fisher Scientific (6 mentions) Rabbit anti gapdh, by Cell Signaling Technology (5 mentions) Pvdf membrane, by Merck Group (4 mentions) Rabbit anti akt, by Cell Signaling Technology (3 mentions) Rabbit anti jnk, by Cell Signaling Technology (3 mentions) Rabbit anti p jnk, by Cell Signaling Technology (3 mentions) Rabbit anti p iκbα, by Cell Signaling Technology (3 mentions) Rabbit anti erk1 2, by Cell Signaling Technology (3 mentions) Rabbit anti p38 mapk, by Cell Signaling Technology (2 mentions) Rabbit anti phospho akt, by Cell Signaling Technology (2 mentions) Rabbit anti phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (2 mentions) Rabbit anti erk, by Cell Signaling Technology (2 mentions) Ecl plus wb detection reagents, by Merck Group (2 mentions) Rabbit anti p ikk α β, by Cell Signaling Technology (2 mentions) Rabbit anti β actin, by Cell Signaling Technology (2 mentions) Ripa lysis buffer, by Beyotime (2 mentions) Gel shift assay system, by Promega (2 mentions) Dna retardation gel, by Thermo Fisher Scientific (2 mentions)

44 protocols using rabbit anti p65

1

Immunoblotting Antibody Validation for IFN-γ Signaling

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The antibodies were listed above for flow cytometry at in vitro coculture assay. For immunoblotting, the following antibodies were used: mouse anti–MHC-I (Santa Cruz Biotechnology, sc-55582), rabbit anti-pSTAT1 (Cell Signaling Technology, 7649), rabbit anti-STAT1 (CST, 14994), rabbit anti-IFNGR1 (Millipore, MABF753), mouse anti–β-actin (Sigma-Aldrich, A5441), rabbit anti–RIG-I (Cell Signaling Technology, 3743), rabbit anti–MDA-5 (Cell Signaling Technology, 5321), rabbit anti-MAVS (Cell Signaling Technology, 3993), rabbit anti-pIRF3 (Cell Signaling Technology, 29047), rabbit anti-IRF3 (Cell Signaling Technology, 4302), rabbit anti-p-p65 (Cell Signaling Technology, 3033), rabbit anti-p65 (Cell Signaling Technology, 8242), rabbit anti-pSTING (Cell Signaling Technology, 72971), and rabbit anti-STING (Cell Signaling Technology, 13647). rabbit anti-p65 (Cell Signaling Technology, 8242) was used for RIP and ChIP, and normal rabbit immunoglobulin G (IgG; Cell Signaling Technology, 2729) was served as a negative control.
Wang Y., Zhao Y., Guo W., Yadav G.S., Bhaskarla C., Wang Z., Wang X., Li S., Wang Y., Chen Y., Pattarayan D., Xie W., Li S., Lu B., Kammula U.S., Zhang M, & Yang D. (2022). Genome-wide gain-of-function screening characterized lncRNA regulators for tumor immune response. Science Advances, 8(49), eadd0005.
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2

Western Blot Antibody Panel for Cell Signaling

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Rabbit anti-ERK1/2 MAPK, rabbit anti-phospho-ERK1/2 MAPK (Thr202/Tyr204), rabbit anti-p38 MAPK, rabbit anti-phospho-p38 MAPK (Thr180/Tyr182), rabbit anti-SAPK/JNK, rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185), rabbit anti-AKT, rabbit anti-phospho-AKT, rabbit anti-p65, rabbit anti-IkB, rabbit anti-MMP-2, rabbit anti-MMP-9, rabbit anti-lamin B1, rabbit anti- -β-catenin were from Cell Signaling Technology (USA). Rabbit anti-phospho-IkB was from Abcam (UK). Horsera-dish peroxidase-labeled goat anti-rabbit IgG, horsera-dish peroxidase-labeled goat anti-mouse IgG, mouse anti-GAPDH, and mouse anti-actin were from Multi-Sciences Biotech Co. Ltd (China). Rabbit anti-SLC8A2 was from MBL International Corporation (Japan).
Qu M., Yu J., Liu H., Ren Y., Ma C., Bu X, & Lan Q. (2017). The Candidate Tumor Suppressor Gene SLC8A2 Inhibits Invasion, Angiogenesis and Growth of Glioblastoma. Molecules and Cells, 40(10), 761-772.
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3

Immunofluorescence Staining of Skin Samples

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Skin samples were embedded in OCT (Tissue Tek) and sectioned to 8 μm. HaCaT cells with AQP3 stable overexpression or knockdown were plated in 24-well plates coated with round coverslips and stimulated with 100ng/ml TNF-α (Cat #300-01, PeproTech) in the presence of calcium. The slides and cells were fixed in 4% formalin for 10 min and washed with PBS 3 times for 10 min. Then we blocked with 5% normal donkey serum in PBS supplemented with 0.3% Triton-X100 for 1 h and incubated with primary antibodies at 4 °C overnight. The next day the appropriate fluorophore-conjugated secondary antibodies were used and 4'-6-diamidino-2-phenylindole (DAPI) was used for nuclei visualization. Following primary antibodies were used: anti-human AQP3 (1:100, Sigma-Aldrich), anti-mouse AQP3 (1:500, Abcam), Rat anti-CD4 (1:100, eBioscience), Rabbit anti-p-p65 (1:200, Cell Signaling), Rabbit anti-p65 (1:100, Cell Signaling). The images were acquired using an Eclipse Ni-U Upright Microscope (Nikon) and 5 randomly selected fields were used to count the positive cells. The number of CD4-positive or nuclear p65-positive cells was counted in at least 3 randomly selected microscopic fields (original magnification, 200×) in each sample to calculate the mean number.
Chen M., Peng Q., Tan Z., Xu S., Wang Y., Wu A., Xiao W., Wang Q., Xie H., Li J., Shi W, & Deng Z. (2023). Targeting Aquaporin-3 Attenuates Skin Inflammation in Rosacea. International Journal of Biological Sciences, 19(16), 5160-5173.
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4

Protein Expression Analysis in Rat Heart

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The heart tissues (0.1 g) from each group of rats were collected and homogenized in a 1 mL protein extraction buffer. The supernatant was collected after centrifugation, and BCA Protein Assay Kit was used to detect protein concentrations. Total protein samples (20 µg) were loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer before subsequent transferal to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were sealed with 5% skimmed milk at 37 °C for 120 min ahead of incubation with the primary antibodies: rabbit β-actin (1:1,000, #4970, Cell Signaling), rabbit anti-cleaved caspase3 (1:1,000, #9662, Cell Signaling), rabbit anti-cleaved caspase9 (1:1,000, #9509, Cell Signaling), rabbit anti-AMPKα1 (1:1,000, #2795, Cell Signaling), rabbit anti-NQO1 (1:1,000, #62262, Cell Signaling), rabbit anti-JNK (1:1,000, #9252, Cell Signaling), rabbit anti-p-JNK (1:1,000, #4668, Cell Signaling), rabbit anti-P65 (1:1,000, #8242, Cell Signaling), rabbit anti-p-P65 (1:1,000, #3033, Cell Signaling) at 4 °C overnight. Subsequently, PVDF membranes were incubated for 60 min at 37 °C with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies. Digital image analysis was conducted with Bio-Rad CFX-96 (Bio-Rad, Hercules, CA, USA) to determine and analyze the density of the bands. β-actin was used as the control.
Zhou Q., Song J., Wang Y, & Lin T. (2020). Remifentanil attenuates cardiac dysfunction, lipid peroxidation and immune disorder in rats with isoproterenol-induced myocardial injury via JNK/NF-KB p65 inhibition. Annals of Translational Medicine, 8(8), 551.
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5

Protein Extraction and Western Blot Analysis

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RIPA lysis buffer (Beyotime, Shanghai, China) was utilized to obtain the total proteins. After being quantified by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA), equal proteins were loaded and electrophoresed on a polyacrylamide gel, and wet-transferred to PVDF membranes (Millipore, Bedford, MA). Then the membranes were kept in 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween 20 (TBST) at indoor temperature for 1.5 h and kept in primary antibody solution with a 1:1000 dilution ratio at 4 °C for 12 h. After being treated with secondary antibody solution with a 1:5000 dilution ratio for 1 h at ambient temperature, the membranes were observed with ECL Plus WB detection reagents (Millipore).
The primary antibodies including mouse anti-phosphorylated (p)-p38, mouse anti-p38, rabbit anti-p-extracellular regulated MAP kinase (p-ERK), rabbit anti-ERK, rabbit anti-p-c-Jun NH2-terminal (p-JNK), rabbit anti-JNK, rabbit anti-β-actin, rabbit anti-p-Syk, rabbit anti-Syk, rabbit anti-p- protein kinase C delta (PKC δ), rabbit anti-PKC δ, rabbit anti-p-IKK α/β, rabbit anti-IKK α, rabbit anti-p-P65, and rabbit anti-P65 were bought from Cell Signaling Technology (Danvers, Massachusetts, USA). The rabbit anti-Histone 3 antibody was bought from Abcam (Cambridge, Massachusetts, USA).
Liu X., Xu Y., Li Y., Pan Y., Zhao S, & Hou Y. (2021). Ferumoxytol-β-glucan Inhibits Melanoma Growth via Interacting with Dectin-1 to Polarize Macrophages into M1 Phenotype. International Journal of Medical Sciences, 18(14), 3125-3139.
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6

Transcription Factor Binding Assay

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EMSAs were conducted using the EMSA kit (Promega) as previously described42 with modifications. A431 keratinocytes were serum-starved for 24-hrs followed by 1-hr treatment with TPA (200 nM) prior to isolation of nuclear extracts. For supershift analyses, nuclear extracts were incubated with 1 μL of either rabbit-IgG (Source), rabbit anti-p65 (Cell Signaling), pre-immune sera10 (link), or anti-K1710 (link) antibodies for 15 minutes at room temperature prior to incubation with 32P-labeled NF-κB oligonucleotide. For competition binding analyses, 50-fold excess non-labeled NF-κB or Oct1 (as a control) oligonucleotide was incubated for 15 minutes at room temperature with nuclear extract prior to incubation with 32P-labeled NF-κB oligonucleotide. Oligonucleotide labeling and gel shift assays were conducted following manufacturer's protocol (Promega, Gel Shift Assay System). Samples were resolved on a 6% DNA retardation gel (Invitrogen) in 0.5 × TBE buffer. Autoradiography was carried out on dried gels using phosphor-screens and phosphorimager.
Hobbs R.P., DePianto D.J., Jacob J.T., Han M.C., Chung B.M., Batazzi A.S., Poll B.G., Guo Y., Han J., Ong S., Zheng W., Taube J.M., Čiháková D., Wan F, & Coulombe P.A. (2015). Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes. Nature genetics, 47(8), 933-938.
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7

Macrophage AhR and NF-κB Regulation

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Primary peritoneal macrophages were plated in 150-mm cell culture dishes. Cells were pre-treated for 1 h with SGA360 (10 µM), followed by LPS (5 ng/ml) for 4 h. Chromatin Immunoprecipitation (ChIP) was performed using SimpleChIP® Plus Enzymatic Chromatin IP kit (Cell Signaling Technology, Danvers, MA) according to manufacturer’s instructions. 8 µg of digested chromatin was incubated overnight at 4°C with the following antibodies: rabbit anti-AHR (Biomol International, Plymouth Meeting, PA), rabbit anti-p65 (Cell Signaling Technology, Danvers, MA), normal rabbit IgG (Santa Cruz Biotechnology, CA). Immunocomplexes were captured with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology). Eluted DNA was purified using spin columns and analyzed by quantitative real-time PCR using primers listed in Table S2.
Muku G.E., Lahoti T.S., Murray I.A., Podolsky M.A., Smith K.J., Hubbard T.D., Kuzu G., Gowda K., Amin S.G, & Perdew G.H. (2017). Ligand-mediated cytoplasmic retention of the Ah receptor inhibits macrophage-mediated acute inflammatory responses. Laboratory investigation; a journal of technical methods and pathology, 97(12), 1471-1487.
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8

Analysis of Ocular Immune Response in Mice

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Eyes from OIR and unexposed control mice were fixed and prepared for 10-µm-cross-sectioning. After fixation with 4% paraformaldehyde, permeabilization with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and blocking with 5% bovine serum albumin, cross-sections were respectively incubated with rabbit anti-p-IκBα, rabbit anti-IκBα (Abcam, Cambridge, MA, USA), rabbit anti-p-p65, rabbit anti-p65, rabbit anti-inducible nitric oxide synthase (iNOS) (Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit anti-Arginase-1 (Arg-1) (GeneTex, Irvine, CA, USA) and then incubated with Alexa Fluor 555 anti-rabbit IgG (Cell Signaling Technology) plus Griffonia simplicifolia Isolectin B4 (GSA-Lectin)-labeled fluorescein isothiocyanate (FITC; Vector Laboratories, Inc., Burlingame, CA, USA), PE-F4/80 (eBioscience, Vienna, Austria) plus FITC-GSA-Lectin (Vector Laboratories), or PE-F4/80 plus Alexa Fluor 488 anti-rabbit IgG (Cell Signaling Technology). After incubation with 4′,6-diamidino-2-phenylindole (Beyotime, Shanghai, China), cross-sections were photographed using fluorescence microscopy (Nikon Instruments, Inc., Melville, New York, USA).
Sui A., Chen X., Demetriades A.M., Shen J., Cai Y., Yao Y., Yao Y., Zhu Y., Shen X, & Xie B. (2020). Inhibiting NF-κB Signaling Activation Reduces Retinal Neovascularization by Promoting a Polarization Shift in Macrophages. Investigative Ophthalmology & Visual Science, 61(6), 4.
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9

Antibody Characterization for Cellular Analysis

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The following antibodies were used: mouse anti-F4/80 (Santa Cruz), rabbit anti-iNOS (Cell Signaling Technology), mouse anti-CD80 (Invitrogen Antibodies), rabbit anti-CD206 (Abcam), rabbit anti-SHP2 (Cell Signaling Technology), rabbit anti-COL2 (Abcam), rabbit anti-COL10 (Abcam), rabbit anti-MMP3 (Proteintech), rabbit anti-COX2 (Cell Signaling Technology), rabbit anti-GAPDH (Cell Signaling Technology), rabbit anti-p-P65 (Cell Signaling Technology), rabbit anti-P65 (Cell Signaling Technology), rabbit anti-β-actin (Cell Signaling Technology), rabbit anti-histone H3 (Cell Signaling Technology), rabbit anti-p-AKT (Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), and rabbit anti-p-IKKα/β (Cell Signaling Technology).
Sun Z., Liu Q., Lv Z., Li J., Xu X., Sun H., Wang M., Sun K., Shi T., Liu Z., Tan G., Yan W., Wu R., Yang Y.X., Ikegawa S., Jiang Q., Sun Y, & Shi D. (2022). Targeting macrophagic SHP2 for ameliorating osteoarthritis via TLR signaling. Acta Pharmaceutica Sinica. B, 12(7), 3073-3084.
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10

Immunostaining of STING, IRF3, and p65

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Cells were cultured and treated on poly-D-lysine-coated round coverslips (BD Biosciences). Cell were fixed with 4% paraformaldehyde/PBS for 15 minutes at 37° C and permeabilized with 0.2% Triton X-100/PBS for 10 minutes at room temperature (RT). After blocking with 10% BSA/PBS for 30 min, the coverslips were incubated with the indicated primary antibodies in 4% BSA/PBS overnight at 4° C in a wet chamber. Immunostaining was performed with rabbit-anti-STING polyclonal (21 (link)), rabbit-anti-IRF3 (Santa Cruz Biotechnology), or rabbit-anti-p65 (Cell Signaling). After washing six times with PBS, the coverslips were incubated with fluorescence conjugated secondary antibody (Alexa Fluor 488-goat anti-rabbit IgG) for 2 h at RT in a wet chamber. After washing six times with PBS, the coverslips were mounted onto glass slides with ProLong Gold antifade reagent (Invitrogen). Images were taken with Leika LSM confocal microscope at the Image Core Facility, University of Miami (Miami, FL).
de Queiroz N.M., Xia T., Konno H, & Barber G.N. (2018). Ovarian Cancer Cells Commonly Exhibit Defective STING Signaling Which Affects Sensitivity to Viral Oncolysis.. Molecular cancer research : MCR, 17(4), 974-986.
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