ROCKII is a substrate of Shp2 that mediates Y722 dephosphorylation. (A) In vitro substrate trapping assay. Wild-type (WT), substrate trapping mutant (DM) flag-Shp2, and control beads, as indicated, were incubated with lysates from pervanadate-treated cells expressing wild-type or Y722F myc-ROCKII. The pulldown proteins were analyzed by Western blotting. (B) Shp2 dephosphorylates ROCKII in vitro. Immunoprecipitated myc-ROCKII protein from pervanadate-treated cells was incubated with increasing amounts of GST-N-del Shp2(PTP) at 30°C for 0–20 min and analyzed by Western blotting. (C) HEK293T cells were transfected with control or Shp2 siRNA for 2 d and replated to FN-coated coverslips for 0.5 or 2 h or trypsinized after 2 h of FN-mediated adhesion. Cells were fixed and subjected to pY722-ROCKII immunofluorescence staining. The percentage of trypsinized cells that were positive in pY722-ROCKII staining is shown below. Bar, 20 μm. (D) Immunofluorescence images of pY722 ROCKII in siRNA transfected HEK293T cells on FN-coated coverslips with TAT-RhoAV14 treatment at the indicated time. (E) Western blot of siRNA transfected cells.