In the present study, the influence of the G6PD mutation was assessed by performing immunoblotting. Western blotting was performed as previously described (Dong et al., 2019 (link); Li et al., 2022 (link); Wang et al., 2022 (link)). Cytoplasmic and nuclear protein extraction was performed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, United States). Equal amounts of cytoplasmic or nuclear extracts were separated on 12% SDS polyacrylamide gels and transferred to PVDF membranes. Blots were probed with primary antibodies against NF-κB p65, β-actin or histone H3 (Cell Signaling Technology, Beverly, MA). Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibody. Visualization was conducted using an ECL peroxidase substrate (Millipore, Germany). The molecular weight of NF-κB p65 and p-p65 is 65 kD, β-actin is 45 kD, and histone H3 is 17 kD.
Primary antibodies against nf κb p65
Manufactured by Cell Signaling Technology
Sourced in United States
Primary antibodies against NF-κB p65 are a laboratory tool used to detect and study the NF-κB p65 protein, a key component of the NF-κB transcription factor complex. These antibodies can be used in various experimental techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to identify and quantify the presence and distribution of NF-κB p65 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ecl peroxidase substrate, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Histone h3, by Abcam (1 mentions)
Proteoextract subcellular proteome extraction kit, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Tlr 4, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
6 protocols using primary antibodies against nf κb p65
1
Western Blot Analysis of G6PD Mutation
2
OLED-induced NF-κB p65 Localization
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RAW264.7 cells (3 × 105 cells/well) were seeded in 6-well plates with cover slips overnight. After OLED irradiation, cells on the cover slips were fixed in cold methanol for 10 min, then permeabilized with 0.1% Triton X-100. After blocking in 5% BSA for 1 h, cells were incubated with primary antibodies against NF-κB p65 (Cell Signaling Technology) at 4°C overnight. The primary antibodies were removed by washing three times with PBS, and the samples were further incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti rabbit IgG; Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature. Finally, the cells were mounted with Vectashield mounting medium, stained with DAPI (Vector Laboratories) for visualization of the nucleus, and photographed under a confocal microscope (Olympus).
3
NF-κB Activation Assay in HK-2 Cells
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HK-2 cells were seeded, incubated, and treated with 0.1% DMSO, TanIIA (5 µM), and/or TNF-α (20 ng/ml) for 24 h. Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 5% BSA for 30 min at room temperature. Primary antibodies against NF-κB p65 (1:100, Cell Signaling Technology, MA, United States) were incubated with cells at 4°C overnight. After washing with PBS three times, an FITC-conjugated secondary antibody was added for 60 min at room temperature. All slides were mounted with DAPI-containing mounting medium and then analyzed with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany)
4
Nuclear and Cytosolic Protein Extraction
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Cytosolic and nuclear protein extracts from BMDMs were prepared using the ProteoExtract® Subcellular Proteome Extraction Kit (Merck) as previously described [12 (link)]. Cytosolic and nuclear proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk, and incubated with primary antibodies against NF-κB p65 (1:1000, Cell Signaling Technology), β-actin (1:10,000), and histone H3 (1:1000, Abcam). Membranes were incubated with HRP-conjugated secondary antibodies (1:10,000), and target protein bands were visualized using an ECL detection kit.
5
Immunofluorescence Analysis of NF-κB and TLR-4 in Monocytes
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Monocytes from EN were plated on the cover slips and cultured at up to 50–60% confluence. After treatment, cells were washed with PBS, and fixed with fresh 4% paraformaldehyde solution for 15 min at room temperature. Cells were then washed twice with PBS, followed by incubation in 10% normal rabbit serum–blocking solution for 20 min at room temperature in a humidified chamber. Cells were incubated with specific primary antibodies against NF-κB p65 (Cell Signaling Technologies, Danvers, MA, USA) for 2 h at room temperature in a humidified chamber. Cells were washed 3 times in PBS and incubated with Alexa Fluor 488–conjugated goat anti-rabbit IgG (Life Technologies) for 45 min at room temperature in a humidified chamber. The cells were then washed in PBS, mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies). Images were acquired using Carl Zeiss LSM-710 confocal microscope with 10 fields of view. Above-mentioned protocol is also followed for TLR-4 surface expression studies with TLR-4 (Life Technologies) and LC-3 (Cell signaling Technologies) specific antibodies.
6
NF-κB p65 Intracellular Localization
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Cells were fixed with 4% formaldehyde and then incubated with 5% goat serum. The intracellular location of NF-κB p65 was determined using primary antibodies against NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA) and FITC-conjugated secondary antibody. Nuclei were counterstained with Hoechst (1 µg/mL). Images were obtained by fluorescent microscopy (OLYMPUS IX 81; Olympus, Tokyo, Japan).
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