2.9. Immunoprecipitation
For IP experiments detecting HMGB1 A Box/CXCR4 binding, the primary macrophages or CD3+ T cells isolated by the differential adherence method were extracted with IP protein lysis solution, and a part of the protein solution was reserved as Input, and the rest protein was added to 40μL Protein A+G agarose beads (Beyotime, p2012) and incubated overnight at 4°C. Then, 1μg of CXCR4 monoclonal antibody (Abcam, ab181020) and 1μg of IgG from the same source as CXCR4 antibody were added and incubated for 3 hours at 4°C. Then 1 mL of IP lysis buffer was used to resuspend the agarose beads, centrifuge at 1000 rcf for 5 min, discard the supernatant, and repeat this step 5 times to fully wash unbound proteins. After last washing step, add 40μL of 1×protein loading buffer to the agarose beads, boiling water bath for 5-10min, SDS-PAGE electrophoresis. The corresponding protein bands were detected using CXCR4 and 6×His (Abcam, ab18184) antibodies.
For IP experiments to detect the binding of HMGB1/CXCL12, a part of the supernatant from MPP+-treated midbrain cells was freeze-dried at low temperature, or a part of serum from PD patients whose serum immunoglobulins were removed by ultrafiltration through a 100kDa ultrafiltration tube as Input. And the rest protein was added to 40μL Protein A+G agarose beads and incubated overnight at 4°C. Then add 1μg of HMGB1 monoclonal antibody and 1μg of IgG from the same source of HMGB1 antibody (Abcam, ab190377) and incubate at 4°C for 3 hours. Then use 1 mL of IP lysis buffer to resuspend the agarose beads, centrifuge at 1000 rcf for 5 min, discard the supernatant, and repeat this step 5 times. After washing, add 40μL of 1×Loading Buffer to the agarose beads, centrifuge the agarose beads to the bottom, take a boiling water bath for 5-10 minutes, and perform SDS-PAGE electrophoresis. The corresponding protein bands were detected using HMGB1 and CXCL12 (ab25117) antibodies.
For the experiment to detect the binding of CXCR4 to HMGB1/CXCL12, the CD14+ monocytes of PD patients obtained by magnetic bead sorting were added to IP lysate to extract the protein, and a part was taken as Input. 1μg of CXCR4 monoclonal antibody and 1μg of IgG from the same source as CXCR4 antibody were addedto the remaining protein solution and incubated at 4°C for 3 hours. Then use 1mL of IP lysis buffer to resuspend the agarose beads, centrifuge at 1000 rcf for 5 min, discard the supernatant, and repeat this step 5 times to fully wash unbound proteins. After washing, add 1×Loading Buffer to the agarose beads, centrifuge the agarose beads to the bottom, take a boiling water bath for 5-10 minutes, and perform SDS-PAGE electrophoresis. The corresponding protein bands were detected using CXCR4, HMGB1 and CXCL12 antibodies.