Anti-Phospho-IKB alpha (S32) NFKBIA Rabbit Monoclonal Antibody

Western blot analysis of P-NFKBIA using anti-P-NFKBIA antibody (P01139).
Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Jurkat whole cell lysates,
Lane 2: human HepG2 whole cell lysates,
Lane 3: human SIHA whole cell lysates,
Lane 4: rat liver tissue lysates,
Lane 5: rat thymus tissue lysates,
Lane 6: mouse liver tissue lysates,
Lane 7: mouse thymus tissue 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-P-NFKBIA antigen affinity purified monoclonal antibody (P01139) at 1:500 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 ECL Plus Western Blotting Substrate (Catalog # AR1196-200) with Tanon 5200 system. A specific band was detected for P-NFKBIA at approximately 39 kDa. The expected band size for P-NFKBIA is at 36 kDa.

Duvelisib and chidamide stabilize IκBα via PI3Kδ and HDAC2 targeting, respectively. A Western blotting analysis was performed to determine the expression levels of drug-targeted proteins corresponding to chidamide (HDAC1, HDAC2, HDAC3, HDAC10) and duvelisib (AKT, p-AKT Ser473) in TMD8, Toledo, and DB cells following gradient concentration treatment with chidamide, duvelisib, or their combination. B Western blotting was utilized to assess the protein expression levels of IKKα/β, p-IKKα/β (Ser176/180), IκBα and p-IκBα (Ser32) in DB cells after treatment with specified drug concentrations. C The effects of duvelisib, PI3Kδ inhibitor (PI3Kδ-IN-15), and PI3Kγ inhibitor (AZ2) on IKK and IκBα protein expression in DB cells were evaluated by western blotting. D Following histone H1.5 protein recruitment, the acetylation status of histone H1.5 and its interaction with IκBα were examined. E A co-immunoprecipitation (co-IP) assay was employed to directly detect the acetylation level of IκBα. In western blotting, DMSO served as the vehicle control, diluted in culture medium to a final concentration of 0.05% (v/v).
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Chidamide treatment inhibits HDAC2 activity and leads to histone H1.5 acetylation. A , B Knockdown of HDAC1, HDAC2, HDAC3, and HDAC10 was performed using siRNA. Following histone H1.5 enrichment, western blotting analysis was conducted to detect histone H1.5 acetylation levels and its interaction with IκBα. ( C ) Mass spectrometry identified five histone H1.5 acetylation sites (A-score >13) after chidamide treatment. D – H Acetylation-deficient mutants were generated by replacing these lysine residues with arginine. Following histone H1.5 enrichment, co-immunoprecipitation (Co-IP) assays were performed to evaluate histone H1.5 acetylation, NF-κB p65 protein levels, and histone H1.5-IκBα interaction. The K67 and K93 mutations significantly reduced acetylation and weakened the histone H1.5-IκBα interaction.
Index in PubMed under a CC BY license. PMID: 41053160