Fig. 4. SHP-2 dephosphorylates Spry1 in vitro. (A) (Top) Phosphorylated HA-Spry1 isolated from transfected HEK293 cells treated with bFGF was incubated alone (lane 2) or with purified GST (lane 3), GST-SHP-2^E76A (lane 4), GST-SHP-2^SH2 (lane 5) or GST-SHP-2^P (lane 6). Lane 1, HA-Spry1 before bFGF addition. Upper blot, immunoblot of products probed with anti-phosphotyrosine to detect phosphorylated HA-Spry1. GST-SHP-2^E76A dephosphorylated HA-Spry1 (lane 4). Lower blot, immunoblot probed with anti-HA to detect all HA-Spry1. Similar results were obtained in three experiments. (Bottom) Tyrosine phosphorylated ERK2-HA was prepared and tested as a SHP-2 substrate as above. Upper blot, immunoblot of ERK2-HA incubated alone (lane 8), with GST (lane 9) or with GST-SHP-2^E76A (lane 10), probed with anti-dpERK to detect phosphorylated ERK2-HA. Tyrosine phosphorylation site detected by anti-dpERK (Y185) was not affected by GST-SHP-2^E76A. Lower blot, same blot reprobed with anti-HA to detect all ERK2-HA. Lane 7, ERK2-HA before bFGF addition. ERK2 and Spry1 have another phosphorylation site near the phosphotyrosine: S50 for Spry1 (data not shown) and T183 for ERK2. Similar results were obtained in two experiments. (B) Experiment as in A (top), except upper blot was probed with anti-pY53.