Effect of Sprouty2 expression of cell responses to EphB2 activation. (a–d) EphB2+iFGFR cells in the absence of iFGFR activator were used to analyze the effect of overexpressing Sprouty2 on collapse (a and b) and segregation (c and d) responses to ephrinB1 (see the Results for an explanation of why EphB2-iFGFR cells were used). (a and b) In collapse assays, EphB2+iFGFR cells were transfected with vector coexpressing Sprouty2 and nuclear-targeted RFP. After the addition of ephrinB1-Fc, >90% of nontransfected cells undergo collapse (arrows), whereas only 27% of Sprouty2/RFP-expressing cells undergo collapse. (c) In control cell segregation assays, EphB2+iFGFR cells were transfected with vector expressing RFP and were mixed with ephrinB1+GFP cells. All RFP-expressing cells cosegregate with nontransfected EphB2 cells. (d and e) Cell segregation assays were performed with EphB2+iFGFR cells transfected with Sprouty2+RFP. Many Sprouty2/RFP-expressing cells remain in or are at the interface of ephrinB2+GFP territory and have thus failed to cosegregate with nontransfected EphB2 cells (quantified by counting the number of cells in e). (f and g) The role of FGFR-induced Sprouty genes in the inhibition of EphB2 activation was analyzed by siRNA knockdown. f shows Western blots, and g shows the quantitations of the increase in phospho-EphB2 normalized to the negative control siRNA. Knockdown with two independent sets of Sprouty2 and Sprouty4 siRNAs leads to an increase in ephrinB1-induced EphB2 phosphorylation in EphB2+iFGFR cells (with iFGFR activator) compared with control siRNA. (e and g) Error bars indicate SEM (n = 4). Pi, phosphorylated.