Anti Streptavidin  

Depleting CD47 on macrophages increases engulfment activity, decreases SIRPα signaling and increases SIRPα affinity.？(A) Stable knockdown of CD47 on THP-1 macrophages to 48% or 13% of wild-type (WT) levels was used to phenocopy anti-CD47 effects. Phagocytosis assays with WT or knockdown (KD) THP-1 cells used IgG-opsonized sheep RBCs (ShRBCs) or IgG-opsonized streptavidin microbeads. Microscopy fields randomly selected and 200 macrophages counted (n=3, mean±s.e.m.). Images, engulfment of ShRBCs. Arrows denote phagocytic events. Scale bar: 10？μm. THP-1 cells with 13% CD47 levels had higher levels of engulfment than WT (？40%). *P<0.03. (B) SIRPα immunoprecipitation from lysates of THP-1 macrophages under basal conditions using anti-SIRPα (SE7C2 clone) and immunoblotted for phospho-tyrosine (pTyr; C-20 clone) (n=3, mean±s.e.m.). The normalized SIRPα pTyr signal increases linearly from zero in relation to the CD47 level. (C) (i) To quantify the effective affinity of CD47 for SIRPα in？trans？on WT and KD THP-1 macrophages, binding of fluorescent soluble CD47 was measured by flow cytometry and normalized to 8？μM data. All data fits？y=A x/(Kd+x) (R2>0.93) for apparent dissociation constants (Kd). (ii)？Kd？increases linearly in relation to the CD47 level, and the non-zero intercept corresponds to the highest affinity for CD47？SIRPα as measured for human RBCs that lack SIRPα (Fig.？1B). Fig 2. PMID:？31964705