PA5-16545

antibody from Invitrogen Antibodies

Targeting: RELA NFKB3, p65

Provider product page for PA5-16545

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [35]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Immunohistochemistry [1]
Other assay [7]

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Product number: PA5-16545 - Provider product page
Provider: Invitrogen Antibodies
Product name: NFkB p65 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: PA5-16545 targets NFkappaB/p65 in IHC (P), IF, and WB applications and shows reactivity with mouse, Rabbit, Rat, Xn, Bovine, Canine, Drosophila, Hamster, Human, and Porcine samples. The PA5-16545 immunogen is a synthetic peptide from the C-terminus of human NFkappaB protein. This antibody was orginally validated as part of a Thermo Scientific Cellomics High Content Screening Kit. The antibody sold separately may have slightly different performance and may need to be further optimized for the best results.
Reactivity: Human, Mouse, Rat, Bovine, Canine, Drosophila, Hamster, Porcine, Rabbit, Xenopus
Host: Rabbit
Isotype: IgG
Vial size: 500 µL
Concentration: 1 mg/mL
Storage: Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

RELA protein structure - PA5-16545 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of RELA was performed with 10 µg of A549 cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against RELA (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. RELA was detected at ~ 65 kDa using RELA Rabbit polyclonal antibody (Product # PA5-16545) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Rabbit (Product # 31461) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). RELA Antibody (Product # PA5-16545) confirms silencing of RELA expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NFkappaB/p65 was performed by loading 50 µg of Jurkat cell lysate per well onto a SDS-PAGE gel. Proteins were transferred to a membrane and blocked with 5% nonfat dry milk in PBS + 0.1% Tween-20 for 30 minutes at room temperature. The membrane was probed with a NFkappaB/p65 polyclonal antibody (Product # PA5-16545) at a concentration of 5 µg/mL for 1 hour at room temperature, washed in PBST, and probed with an anti-rabbit IgG-HRP secondary antibody at a dilution of 1:2500 for 1 hour at room temperature. Detection was performed using a chemiluminescent substrate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Radiation-induced DNA damage and subsequent cellular senescence in-vitro experiments with rat glomerular endothelial cells. ( a - f ) Radiation induced DNA double strand breaks (DSBs) in glomerular endothelial cells, shown as gammaH2AX (a DSBs marker). (green, gammaH2AX and bleu, nuclei). ( g , h , Bar, 50 um) Representative images of SA-beta-gal staining in cultured glomerular endothelial cells at Days 0 and 20. ( i ) Irradiated glomerular endothelial cells displayed increase in positive staining for SA-beta-gal in a time-dependent manner, at Days 3, 10 and 20. ( j ) Irradiated cells showed positive staining for p53 in the nucleus. ( k ) Irradiated cells sustained higher expression level of p21 mRNA compared to normal cells, at Days 3, 10, and 20. ( l ) Irradiated cells showed higher expression levels of p16 mRNA at Day 20. ( m - q ) SASP (IL-6, TNF-alpha, VEGF-A, ICAM and VCAM) was increased in irradiated glomerular endothelial cells at Days 15 and 20. ( r - t , Bar 100 um) Irradiated cells showed increase of expression level of mRNA. Additionally, they showed activation of NF-kB revealed by positive staining in nuclear localization and phosphorylated IkappaBalpha. Values are expressed as mean +- SD. *P < 0.05, **P < 0.01, and **P < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Inflammatory cytokines. a Immunohistochemistry staining for inflammatory markers TNF-alpha and IL-6 in kidney tissue. b Western blot on kidney tissue measuring inflammatory markers TNF-alpha and NF-kappaB (left), alongside their quantification (right). c Quantitative real-time PCR on kidney tissue measuring inflammatory markers Nfkb , Il6 , and Tnfa . d ELISA measurement of blood serum concentrations of cytokines IL-1beta, IL-6, and TNF-alpha. Each group has n = 5 mice. Significant difference a p < 0.05: relative to untreated control; b p < 0.05: relative to AKI; c p < 0.05: relative to AKI-EV; d p < 0.05: relative to AKI-EV-pFUS

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Saururus chinensis (SC) water-extract controlled inflammation through the NF-kappa B/COX-2 and PGE 2 axis. (A-P) Saururus chinensis (SC) water-extract inhibited COX-2 expression via preventing NF-kappa B translocation. Compared to the results in ovalbumin and fine dust treatment group in tap water treatment group the expression of COX-2 protein (red spot) was decreased significantly. In ovalbumin and fine dust treatment group NF-kappa B proteins were translocated from cytoplasm to nucleus and the expression of COX-2 protein was increased effectively. Both in dexamethasone treatment group and in 300 mg/kg SC treatment group, the proteins expression were down-regulated, both of NF-kappa B and COX-2. (R-I) SC effectively inhibited the PGE 2 expression. R, tap water treatment (control) group; S, ovalbumin and fine dust treatment (induction) group; T, ovalbumin, fine dust, and dexamethasone treatment (positive control) group; I, ovalbumin, fine dust, and 300 mg/kg SC treatment group. Arrow, NF-kappa B; arrowhead, COX-2. Magnification, 1000 x. Scale bar, 50 mu m. (Q) The quantitative score of NF- kappa B p65 vs. nucleus or COX-2 vs. nucleus. All the values are presented as mean +- standard deviation. n = 8 per each group. * p < 0.05 vs. tap water treatment group; $ p < 0.05 vs. ovalbumin and fine dust treatment group; $$ p < 0.001 vs. ovalbumin and fine dust treatment group. (V) The number of PGE 2 positive cells in the lung tissue. All values are presented as mean +- stan

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Effects of capsaicin and LPS treatment on the phosphorylation of NF-kappaB p65 in LPS-challenged IPEC-J2 cells. The level of phosphorylated NF-kappaB p65 was evaluated using Immunofluorescence staining (A) and Western blotting (B) , respectively. For Immunofluorescence staining, IPEC-J2 cells were seeded into coverslips at a density of 1x10 5 /well and cultured for 2 weeks. After being pretreated with capsaicin (100 muM), cells were stimulated with LPS. Cells then were then fixed for phosphorylated NF-kappaB p65 staining as described in the Materials and Methods. As for Western blot, IPEC-J2 cells were cultured in 6-well plates. Cells were firstly pre-treated with capsaicin (100 muM) for 2 h and then stimulated with LPS (10 mug/mL) for 6 h. The protein was extracted and the total NF-kappaB p65, as well as phosphorylation of NF-kappaB p65 in LPS-challenged IPEC-J2 cells were detected as described in the Materials and Methods. Values are presented as mean +- SEM. Different letters on bars (a, b) indicate significant differences, P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 NF-kappaB rapidly and transiently translocates to the nucleus following tail amputation, and this requires active NADPH oxidase (A-C) Western blots showing RelA in cytoplasmic (c) and nuclear (n) protein fractions from stage 55 tail sections as drawn, at indicated hours post amputation (hpa). Arrows indicate approximate site of cuts. Non-specific (NS) bands for each fraction are included as loading controls. (A) Untreated tadpoles (B) Tadpoles treated with 2 muM DPI from 1 h before amputation, to disrupt ROS production. (C) Graph showing percentage of tadpoles regenerating tails after 7 days, following chemical inhibition of NADPH oxidase (Nox) with 2 muM DPI treatment starting 1 h before amputation of the distal third of stage 43 tadpole tail, or 0.1% DMSO vehicle. Single cohort of tadpoles with N = 14-15 tadpoles per replicate Petri dish; points represent three replicates for each time point. Data were analyzed by two-way ANOVA followed by Sidak's multiple comparisons of all means; adjusted p values *p < 0.05, **p < 0.01 are shown for treatments that differ significantly from the equivalent vehicle control. (C') Bar graphs indicate the distribution of phenotypes observed in (C). Raw data can be found in Data S1 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 C. longa and A. hookeri co-treatment inhibits NF-kappaB and COX-2 expression. ( A ) NF-kappaB is downregulated in the nucleus and COX-2 in the cytoplasm. NF-kappaB and COX-2 are not expressed in the control group and only the nucleus is stained with DAPI (blue spots). Carrageenan stimulates the expression of NF-kappaB in the nucleus and that of COX-2 in the cytoplasm. MSM suppresses the expression of NF-kappaB and COX-2 induced by carrageenan, but the expression of NF-kappaB (arrow) and COX-2 (arrowhead) observed is minimal. C. longa and A. hookeri co-treatment significantly inhibits the expression of NF-kappaB and COX-2. ( B ) The graph and scores present the immunofluorescent results using the image analyzing program in the K1-Fluo confocal microscope. MSM and C. longa and A. hookeri co-treatment statistically significantly control NF-kappaB and COX-2 expression effectively. ( C ) Compared to the density of NF-kappaB in the nucleus and cytoplasm, the pattern is similar to that observed in the immunofluorescence assay. C. longa and A. hookeri co-treatment blocks the translocation of NF-kappaB into the nucleus from the cytoplasm and inhibits the expression of COX-2 in the cytoplasm. ( D ) Depending on the image analysis of the western blotting of NF-kappaB (in nucleus and in cytoplasm) and COX-2 (in the skin tissue), the density graph based on each band per beta-actin was presented using the Image J program. ( E ) MSM and C. longa and A. hookeri co

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 FIGURE NF-kappaB is activated and participates in the AKIP1 mediated induction of the EMT. A, The cytoplasmic and nuclear proteins in Lv-AKIP1-HeLa, Lv-AKIP1-CaSki, and control LEV cells were extracted. Levels of indicated proteins were measured using western blot. B, Measurement of protein signal intensities using ImageJ software. C, AKIP1-HeLa and Lv-AKIP1-CaSki cells were transfected with siRNA-negative control (siNC) or p65 siRNA transiently for 72 hours to knock down p65. The expression levels of AKIP1, p65, E-cadherin, and fibronectin proteins were detected using western blot analysis. D, Measurement of protein signal intensities was done using ImageJ software. E, Knockdown of p65 using siRNA in Hela and CaSki cells was confirmed using RT-PCR. Mean +- SD is used for the description of data from three independent measurements. ** indicates P < .01, as compared to the control