Rabbit anti nf kb p65

Fresh skin tissues from mice were embedded with OCT (Tissue Tek). Skin sections (6 μm) and HaCaT cells grown on round glass coverslips in 24-well plates were fixed with 4 % paraformaldehyde (PBS) for 10 min. Sections were blocked with 5 % donkey serum for 1 h after permeabilization with 0.3 % Triton X-100 for 15 min at room temperature. Primary antibodies were incubated overnight at 4 °C. After washing with PBS, Alexa Fluor 488-conjugated or 594-conjugated secondary antibody (Thermo Fisher Scientific) was added for 1 h at room temperature. Sections were washed and counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). All images were captured with a Nikon DS-Ri2 (Nikon, Japan). The following primary antibodies were used: Rat anti-CD4 (1:100, eBioscience), Rat anti-CD31 (1:100, eBioscience), Rabbit anti-Ki67 (SolA15) (1:500, eBioscience™, #PA5-114437), Rabbit anti-NF-kB p65 (1:100, Cell Signaling) antibodies.

Cells were seeded and incubated overnight in culture media. Cells were lysed in Laemmli buffer supplemented with beta-mercaptoethanol and HALT protease and phosphatase inhibitor cocktail (Sigma). Isolation of nuclei from whole-cell lysis adapted a standard centrifugation-based protocol (79 (link)). Briefly, cells were lysed in 0.1% Igepal in PBS supplemented with phosphatase inhibitor cocktail. Nuclear fractions were sedimented by centrifugation. Whole-cell lysates and nuclear fraction were resolved using SDS-PAGE. Western blot was done using a standard protocol, and images were processed and quantified using ImageJ (https://imagej.net/ij/download.html). For phospho-NF-kB experiments cells were serum starved for 4 to 8 h before lysing. Recombinant human TNF-α (R&D systems 210-TA) treatment was done at 20 ng/ml for 15 min before lysing cells. Recombinant human IFN-β (R&D systems 8499-IF-010) was done at 1000 IU/ml for 24 h before lysing cells. Antibodies used include: rabbit anti-JADE3 (Abcam #129495) used at 1:750 to 1:1000, rabbit anti-IFITM3 (Cell Signaling #59212) used at 1:750 to 1:1000, rabbit anti-NF-kB p65 (Cell Signaling #8242) used at 1:1000, rabbit anti-phospho-NF-kB p65 Ser536 (Cell Signaling #3033) used at 1:1000, rabbit anti-H3 (Cell Signaling), anti-Flag horseradish peroxidase (HRP) (Sigma), anti-rabbit IGG HRP (Sigma), and anti-GAPDH HRP (Sigma).