Figure 5.

Spry2 prevents relocalization of a DAG reporter protein to the plasma membrane and inhibits signaling downstream of DAG. (A) Fluorescent images of RBL-2H3 mast cells transfected with a DAG binding reporter protein construct (PKCγ-C1-GFP) without (a and a′) or with (b and b′) transfection of Spry2 before (a and b) and after (a′ and b′) cross-linking of the IgE receptors with DNP-BSA. c and c′ show DsRed Spry2 expression in the mast cell presented in b and b′. (B) The mast cell from b and b′ was treated with PMA showing membrane localization of DsRed Spry2 and PKCγ-C1-GFP after stimulation. Sixteen vector-transfected cells and 13 Spry2-transfected cells were analyzed as described above with results presented in Table 1. (C) NIH 3T3 cells engineered to express Spry1 under doxycycline control treated overnight with doxycycline in starvation media, treated with PDGF BB (20 ng/ml) for the indicated times, and then cell lysates were immunoblotted with antibodies directed against PKCδ and phospho-threonine 505-modified PKCδ. (D) Sprouty1,2,4 null or control fibroblasts were serum starved and treated with PDGF BB (20 ng/ml) for the indicated times. Cell lysates were immunoblotted for PKCδ and phospho-threonine 505-PKCδ. WT, wild type.