The DNA-binding ability of p50 is increased in Lrrk2-KD microglia in response to LPS stimulation.

<p>(<b>A</b>) The DNA-binding activity of NF-κB was analyzed by EMSA. Nuclear extracts were prepared from control and Lrrk2-KD cells at the indicated times after LPS treatment. Specific binding was analyzed using an excess (20×) of unlabeled (Cold) consensus NF-κB sequence. Two specific NF-κB-DNA complex bands (arrowhead and arrow) were detected. (<b>B</b>) A supershift assays were performed using NF-κB p50 and p65 antibodies. Nuclear extracts obtained 30 min after LPS treatment were preincubated with antibodies. Arrows and arrowheads in (A, B) indicate p50/p50 complex and p50/p65 complex, respectively. (<b>C</b>) DNA affinity precipitation assays were performed using nuclear extracts prepared from untreated and LPS-treated control and Lrrk2-KD microglia. Nuclear extracts (100 µg protein) were incubated with biotin-labeled NF-κB consensus sequence, and then precipitated with streptavidin-conjugated agarose beads. The amount of p50 or p65 in the nuclear extracts (nuclear extract), and bound to DNA (DNA-binding) were analyzed by Western blotting. TBP was used as a nuclear marker. The arrowhead in (C) indicates p50. Data are representative of three independent experiments.