Anti-NFkB/NFKB1 p105/p50 Antibody Picoband®

FLIP activates the canonical NF-κB pathway in myeloid cells. a Suppressive activity of THP1 cells transfected with c-FLIP RNA was measured by enumeration of an absolute number of human CD3 + T cells collected after in vitro co-culture. b Immunofluorescence confocal microscopy of p65 (red), p50 and p52 (green) nuclear translocation in transfected THP1 cells. c Nuclear c-FLIP protein expression by western blot in THP1 cells transfected with either GFP or c-FLIP RNA at different time points. d Immunofluorescence confocal microscopy of p50 (red) and c-FLIP (green) nuclear translocation in transfected THP1 cells. e CD11b + Ly6C + cells (M-MDSCs) were isolated from the spleen of MCA203 tumor-bearing, wild-type mice by flow sorter and transfected for 18 h with scramble, IKKα, IKKβ, or the combination IKKα plus IKKβ siRNAs. After transfection, cells were washed three times. M-MDSCs were co-incubated with peptide-stimulated CellTrace-labeled OT-I cells. Suppressive activity was measured by enumerating absolute numbers of CD8 + T cells collected after in vitro co-culture. f PD-L1 expression in transfected M-MDSCs. Data are presented either as mean ± s.e.m of three independent experiments ( a ) or as mean ± s.e.m of four independent experimental transfections ( e , f ) where each plot refers to CD11b + Ly6C + cells isolated from the pooled spleens of three tumor-bearing mice. Original images × 800 for all panels ( b , d ). * P < 0.05, ** P < 0.01; *** P < 0.001; n.s., not significant, by Mann–Whitney test ( a , b , e , f )
Index in PubMed under a CC BY license. PMID: 30518925

Flow Cytometry analysis of Hela cells using anti-NFkB p105/p50/NFKB1 antibody (PB9149).
Overlay histogram showing Hela cells stained with PB9149 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-NFkB p105/p50/NFKB1 Antibody (PB9149, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Western blot analysis of NFkB p105/p50/NFKB1 using anti-NFkB p105/p50/NFKB1 antibody (PB9149).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Raji whole cell lysates,
Lane 2: human SH-SY5Y whole cell lysates,
Lane 3: human Hela whole cell lysates,
Lane 4: human Jurkat whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-NFkB p105/p50/NFKB1 antigen affinity purified polyclonal antibody (Catalog # PB9149) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for NFkB p105/p50/NFKB1 at approximately 50 kDa, 120 kDa. The expected band size for NFkB p105/p50/NFKB1 is at 105 kDa.

IF analysis of NFkB p105/p50/NFKB1 using anti-NFkB p105/p50/NFKB1 antibody (PB9149).
NFkB p105/p50/NFKB1 was detected in an immunocytochemical section of U2OS cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-NFkB p105/p50/NFKB1 Antibody (PB9149) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.