SIRPα on activated monocytes and macrophages forms clusters/complexes.

<p>A) Assessing SIRPα on naïve PBMC, transmigrated monocytes, PMA-induced THP-1 and murine macrophages by immunofluorescence staining using anti-SIRPα.ex and mAb P84, an antibody specific for the murine SIRPα extracellular domain. B) Staining of SIRPα and CD47 on PMA-stimulated THP-1 using anti-SIRPα.ex and mAb B6H12.2. C) DTSSP crosslinking to assess protein complexes consisting SIRPα. Different cells were treated with DTSSP (25°C, 30 min) prior to lysis in detergent-containing buffer. Lysate samples were analyzed by SDS-PAGE and WB under non-reducing or reducing (+DTT) conditions. The latter reverses the DTSSP crosslinking. Note: murine SIRPα appears to be 110-120 kD, larger than the human counterpart. Dot blot detection of SIRPα was performed by directly absorbing cell lysates onto nitrocellulose, prior to antibody blotting. D) Equilibrium centrifugation analysis of SIRPα complexes. After DTSSP crosslinking, cell lysates were applied to ultracentrifugation in sucrose density gradients. Following fractionation, SIRPα localization was analyzed by WB and compared to the sedimentation of a reference protein marker.