Antibody-induced heterodimerization of the cytoplasmic domains of _IL-3 and β_IL-3 induces tyrosine phosphorylation of JAK-2. Factor- and serum-starved cells were incubated on ice with anti-CD16 antibodies for 10 minutes (16). Cells were washed once with RPMI, and after the addition of secondary goatmouse, the cells were stimulated at 37°C for 10 minutes. Alternatively, the cells were stimulated with IL-3 for 10 minutes or left unstimulated as a control (−). Cell lysates were subjected to immunoprecipitation (IP) with antibodies against JAK-2, and the eluates were resolved by SDS-PAGE. The membranes were immunoblotted (IB) with 4G10 (PY) to detect phosphorylation on tyrosine.