- 710450 - Invitrogen Antibodies NFKB1 antibody

Submitted references Regulatory role of DEPTOR‑mediated cellular autophagy and mitochondrial reactive oxygen species in angiogenesis in multiple myeloma.

Wang J, Chen J, Qiu D, Zeng Z
International journal of molecular medicine 2021 Feb;47(2):643-658

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NFkB p50 in whole cell extracts from HeLa using a NFkB p50 Recombinant Rabbit Polyclonal Antibody (Product # 710450) at a dilution of 2 µg/mL. Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody followed by chemiluminescence (ECL). Results show a band at ~50kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NFkB p50 was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with NFkB p50 (24HCLC), Recombinant Rabbit Polyclonal Antibody (Product # 710450) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of NFkB p50 in HeLa cells using a NFkB p50 Recombinant Rabbit Polyclonal Antibody (Product # 710450) followed by detection using an Alexa Fluor 488-conjugated Goat anti-Rabbit secondary antibody (green) (Image A). Nuclei were stained using DAPI (Image B) and actin stained with Alexa Fluor 594 phalloidin (red) (image C). Image D is a composite image showing nuclear localization of p50.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of NFkB p50 showing staining in the cytoplasm of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with NFkB p50 Recombinant Rabbit Polyclonal Antibody (Product # 710450) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of NFkB p50 showing staining in the cytoplasm and nucleus of paraffin-embedded human tonsil tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with NFkB p50 Recombinant Rabbit Polyclonal Antibody (Product # 710450) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of NFkB p50 showing staining in the cytoplasm and nucleus of paraffin-embedded mouse spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a NFkB p50 Recombinant Rabbit Polyclonal Antibody (Product # 710450) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Enrichment of endogenous NFkB p50 protein at specific gene loci using Anti-NFkB p50 Recombinant Rabbit Polyclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-NFkB p50 Recombinant Rabbit Polyclonal Antibody (Product # 710450, 5 µg) on sheared chromatin from 2 million HeLa cells treated with 50 ng/mL of TNFalpha for 45 minutes using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoter of active IL-8, IL-6 gene, used as positive control target, and the GAPDH, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Mitochondrial damage signals in DEPTOR-inhibited RPMI8226 and MM.1S cells. RPMI8226 and MM.1s cells were infected with lentiviral GFP and DEP3 for 5 days. (A and B) The mtROS levels of the RPMI8226 and MM.1S cells in the indicated groups stained with the MitoSOX probe was evaluated using a fluorescence microscope (scale bar, 10 u m) and flow cytometric analysis, respectively. (C) The NF-kappaB activity of RPMI8226 or MM.1S cells in the indicated groups was detected by luciferase assays. Cells were co-transfected with pNF-kappaB-TA-luc with Renilla luciferase reporter (as an internal control) for 24 h and treated with GFP or DEP3 for 48 h. (D-G) The mitochondrial and cytoplasmic fractions of the cultured cells were separated using the Mitochondria/Cytosol Fractionation kit. Protein expression of Cyt c in the (D) mitochondria and (E) cytoplasm of the indicated groups for RPMI8226 cells was examined by western blot analysis. Primary antibodies with COXIV and beta-actin was used as the mitochondrial and cytosolic loading control, respectively. Protein expression of Cyt c in the (F) mitochondria and (G) cytoplasm of the indicated groups for MM.1S cells was examined by western blot analysis. Primary antibodies with COXIV and beta-actin was used as the mitochondrial and cytosolic loading control, respectively. Protein expression levels of DEPTOR and IkappaB-alpha in the cytoplasm of the indicated groups for (H) RPMI8226 and (I) MM.1S cells were examined by western blot anal

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Autophagy inducer and mitochondrial-specific antioxidants reversed the effects of DEPTOR on the angiogenesis of MM cells. RPMI 8226 and MM.1S cells were transfected with lentivirus DEP3 for 5 days and exposed to SMER28 (10 u M) or Mito-TEMPO (10 u M) for 24 h. (A-D) The mitochondrial and cytoplasmic fractions of the cultured cells were separated using the Mitochondria/Cytosol Fractionation kit. Protein expression of Cyt c in the (A) mitochondria and (B) cytoplasm of the indicated groups for RPMI8226 cells was examined by western blot analysis, respectively. Primary antibodies with COXIV and beta-actin was used as the mitochondrial and cytosolic loading control, respectively. Protein expression of Cyt c in the (C) mitochondria and (D) cytoplasm of the indicated groups for MM.1S was examined by western blot analysis, respectively. Primary antibodies with COXIV and beta-actin was used as the mitochondrial and cytosolic loading control, respectively. Protein expression levels of DEPTOR and IkappaB-alpha in the cytoplasm of the indicated groups for (E) RPMI8226 and (F) MM.1S cells were examined by western blot analysis, respectively. (G-L) The nuclear and cytoplasmic protein fractions of the cultured cells were isolated using the NE-PER(tm) Nuclear and Cytoplasmic Extraction Reagents. Protein expressions of (G) p-p50 and p-p65 in the nuclear, (H) p50 and p65 in the whole cell lysates of the indicated groups for RPMI8226 cells was examined by western blot analysis, respect

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