nf-kb p65 binding activity assay kit  (Active Motif)

Journal: Pharmaceutics

Article Title: CuMV VLPs Containing the RBM from SARS-CoV-2 Spike Protein Drive Dendritic Cell Activation and Th1 Polarization

doi: 10.3390/pharmaceutics15030825

Figure Lengend Snippet: VLPs induce NF- k B nuclear translocation in human DCs. DCs were treated with 20 μg/mL Poly I:C and 2.5 μg/mL R848, or 10 μg/mL VLPs, or VLPs plus 20 μg/mL Poly I:C and 2.5 μg/mL for 24 h. ( A ) I k Bα levels were evaluated using Western blot. Each column expresses the normalized expression to the control (not stimulated cells) and represents the mean ± SEM of five independent experiments. Representative images are shown. **** p < 0.0001 relative to the CTR. ( B ) Fluorescence staining of the nuclei (blue) and immunofluorescence staining of NF-κB/p65 (green) were performed as described in Materials and Methods. Control cells (CTR) were not stimulated. Scale bar 10 µm. Representative images of each condition are shown. LPS: lipopolysaccharide; VLP: virus-like particles, PolyI:C: polyinosinic:polycytidylic acid, R848: resiquimod.

Article Snippet: Then, wells were washed once with PBS, and DCs were fixed with 4% paraformaldehyde for 15 min at room temperature and washed three times with PBS 1% + glycine 0.1 M. Cells were then blocked for 1 h with 5% goat serum + 0.3% Triton X-100 in PBS and further incubated with the primary rabbit monoclonal anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) p65 XP ® antibody, at the dilution of 1:400 (Cell Signaling Technology, Inc., Danvers, MA, USA), overnight at 4 °C.

Techniques: Translocation Assay, Western Blot, Expressing, Control, Fluorescence, Staining, Immunofluorescence, Virus