NF-κB p65 nuclear translocation studies by ELISA

The levels of monocyte p65 were measured to investigate the effects of baicalin pretreatment on H. parasuis-activated monocytes NF-κB signaling. The cells of 1 × 10⁷ seeded on 6-well plates were pretreated with baicalin (25, 50, 100 μg/mL) for 2 h. Then 1.0 × 10⁷ CFU/mL H. parasuis was added into the wells. After stimulation of 6 h, the cells were collected and cytoplasmic protein and nucleoprotein were extracted with a cytosolic—nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China). Protein concentrations were determined with the bicinchoninic acid (BCA) protein assay reagents (Beyotime Biotechnology) according to the manufacturer’s instructions. The expression of NF-κB p65 in cytoplasmic protein and nucleoprotein was measured by NF-κB ELISA Kit (Blue Gene Biotechnology, Shanghai, China) and the results of nuclear translocation of NF-κB p65 are presented by nuclear protein expression of NF-κB p65/cytoplasmic protein expression of NF-κB p65.