Fig. 3. SHP-2 forms a complex with Spry1. (A) Co-immunoprecipitation analysis. HEK293 cells were transfected with plasmids expressing HA-Spry1 (lanes 1-3, 7-9) or HA-Spry1^Y53F (lanes 4-6), plasmid expressing SHP-2-V5 (lanes 1-6), and empty vector (lanes 1, 4, 7) or vector expressing FGFR3 (lanes 2, 5, 8) or constitutively-active FGFR3^K644E (lanes 3, 6, 9) to increase FGF pathway activity. SHP-2-V5 was immunoprecipitated from cell lysates. (Top) Immunoblot of immunoprecipitates probed with anti-HA to detect co-immunoprecipitated HA-Spry1. (Middle) Blot reprobed with anti-V5 showing SHP-2-V5 in immunoprecipitates. (Bottom) Immunoblot of whole cell lysates probed with anti-HA to show HA-Spry1 expression. Similar results were obtained in three experiments. (B) GST pull-down analysis of SHP-2 interaction with Spry1. HEK293 cells were transfected with plasmids expressing HA-Spry1 and FGFR3^K644E to increase HA-Spry1 phosphorylation. Cell lysates were incubated with beads coated with purified GST (lane 1), or GST fused to constitutively active SHP-2^E76A (lane 2) or to truncated SHP-2 containing only the SH2 domains (lane 3). (Top) Immunoblot of proteins bound to beads, probed with anti-HA. HA-Spry1 bound to GST-SHP-2^E76A (lane 2) and, less well, to GST-SHP-2^SH2 (lane 3). Similar results were obtained in three experiments. (Bottom) Coomassie Blue-stained SDS-PAGE gel of purified GST fusion proteins. Positions of full-length proteins are indicated; lower molecular weight forms are presumably breakdown products.