Fig. 6. Wild-type (wt) FcRIIA but not Y298F FcRIIA mediates spreading of BHK transfectants. (A) Cells were plated on coverslips coated with IV.3 anti-FcRII. In control samples, BSA or fibronectin (Fn) were used as a substratum. Where indicated, cells were pretreated with 15 µM cytochalasin B (CB), 10 µM PP1, 1 mM HMA or 8 mM MCDX. Cells after 2 hours (or 5 hours, as indicated) of spreading are shown. Organization of actin filaments visualized with phalloidin-TRITC is demonstrated in the lower panel. Bars, 20 µm. (B) Quantification of spreading of BHK cells transfected with wild-type FcRIIA and Y298F FcRIIA on IV.3-coated substratum 2 hours after plating. Spreading of cells transfected with wild-type receptor was inhibited after pretreatment with 15 µM cytochalasin B, 10 µM PP1, 1 mM HMA or 8 mM MCDX. The results are the means±s.e.m. from two experiments. (C) Tyrosine-phosphorylated FcRIIA is marked by an arrowhead. Phosphorylation of wild-type FcRIIA was induced by spreading of BHK cells on IV.3 but not on fibronectin-coated substratum. No phosphorylation of Y298F FcRIIA was detected during spreading of the receptor-expressing cells on IV.3-coated substratum. Cell spreading was carried out for 0.5, 2 and 3 hours. The blot was reprobed with anti-actin to reveal equal loading of proteins. Molecular mass standards are shown on the left in kDa.