Anti-NAK/TBK1 Antibody Picoband®

Western blot analysis of NAK/TBK1 using anti-NAK/TBK1 antibody (A00261-1).
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 293T whole cell lysates,
Lane 2: human HepG2 whole cell lysates,
Lane 3: human LNCAP whole cell lysates,
Lane 4: rat brain tissue lysates,
Lane 5: rat C6 whole cell lysates,
Lane 7: mouse brain tissue lysates,
Lane 8: mouse Neuro-2a 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-NAK/TBK1 antigen affinity purified polyclonal antibody (Catalog # A00261-1) at 0.25 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 NAK/TBK1 at approximately 84 kDa. The expected band size for NAK/TBK1 is at 84 kDa.

HMGCR inhibition combined with radiotherapy significantly activates the cGAS–STING pathway. a , A volcano plot of differentially expressed genes between the combination group and the radiotherapy group. b GSEA of the TCR signaling pathway (KEGG: MMU04660) and T-cell-mediated immunity (GO: 0002456) between the combination therapy group and the radiotherapy group. c A heatmap of log 2 FC to depict the gene expression associated with type I IFN. d The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein extracted from CT26 tumors was detected by western blot. e , The relative expression of CCL5, CXCL10 and IFNβ mRNA extracted from CT26 tumors was detected by qPCR. f , Representative IF images of p-TBK1 and p-IRF3. g , Representative IHC images of IFN-β. h , The levels of IFN-β, CCL5 and IFN-γ protein in tumor tissues were measured via ELISA. Scale bar, 50 μm. ** P < 0.01; * P < 0.05.
Index in PubMed under a CC BY license. PMID: 40355720

Cholesterol impairs radiotherapy-induced cGAS–STING activation and lovastatin rescues this activation in vitro. a The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein extracted from CT26 cells with different treatments (ctrl, cholesterol 50 μM, MβCD 2 mM, IR 6 Gy, IR + cholesterol and IR + MβCD) and detected by western blot. b The relative expression of CCL5, CXCL10 and IFNβ mRNA extracted from CT26 cells was detected by qPCR. c The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein was extracted from HCT116 cells with different treatments (ctrl, cholesterol 50 μM, MβCD 2 mM, IR 6 Gy, IR + cholesterol and IR + MβCD) and detected by western blot. d The relative expression of CCL5, CXCL10 and IFNβ mRNA extracted from HCT116 cells was detected by qPCR. e , f The expression of p-TBK1, p-IRF3, TBK1 and IRF3 protein extracted from CT26 cells and HCT116 cells with different treatments (ctrl, lovastatin 10 μM, IR 6 Gy and IR + lovastatin) was detected by western blot ( e ) and quantitative anslysis ( f ). g , h , Confocal fluorescence microscopy was conducted on CT26 ( g ) and HCT116 ( h ) cells with different treatments. The cells were labeled with DAPI (blue) and p-TBK1 (green) or p-IRF3 (green). i The levels of IFN-β and IFN-γ in the supernatant of co-cultures of MC38-OVA cells and OT-1 mouse spleen cells as well as HCT116 cells and human PBMCs were measured using ELISA. The ratio of immune cells to tumor cells is 10:1. The co-cultures were maintained for 36 h. j LDH release assay was performed using the supernatants from co-cultures of MC38-OVA cells and OT-1 mouse spleen cells as well as HCT116 cells and human PBMCs. The ratio of immune cells to tumor cells is 10:1. The co-cultures were maintained for 36 h. Scale bar, 5 μm. ** P < 0.01; * P < 0.05; ns, not significant.
Index in PubMed under a CC BY license. PMID: 40355720

IHC analysis of NAK/TBK1 using anti-NAK/TBK1 antibody (A00261-1).
NAK/TBK1 was detected in a paraffin-embedded section of mouse testis tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-NAK/TBK1 Antibody (A00261-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.