Figure 7.

Antiphosphotyrosine staining of FcγR-transfected COS cells after IgG-opsonized particle binding. Transfected COS cells were incubated with IgG-opsonized RBCs for 10 min at 37°C and were then fixed and stained with antiphosphotyrosine mAb 4G10 followed by a secondary FITC-conjugated donkey anti–mouse IgG for detection. A and C show anti-phosphotyrosine staining, whereas B and D show companion nonconfocal phase contrast sections. In A, an FcγRIIA-expressing cell displays tyrosine phosphoprotein accumulation at sites of IgG-opsonized particle binding, indicating triggered tyrosine kinase activity. In B, several cells not expressing FcγR (n, nuclei) can be seen that show only background staining for phosphotyrosine in A, confirming specificity. C and D show an FcγRIA-expressing cell that has bound many IgG-opsonized RBCs, but does not show any phosphotyrosine staining above background levels. Bar, 10 μm.