Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.