2.12. Co-IP analysis

Co-IP was performed according to the method described previously²⁰. In brief, cells were homogenized in RIPA lysis buffer (Weak). Protein concentration was determined using a BCA kit. An equal amount of protein was incubated with anti-Flag magnetic beads overnight. The beads, including the protein that was pulled down, were washed with IP lysis buffer, denatured in 1 × sample loading buffer, and separated by SDS-PAGE. Clean-Blot IP Detection Kit was used to detect functional primary antibodies, HMGB1 and HMGB2.