FIGURE 8.

Catalytic activity and subcellular localization of monoubiquitinated ΔSH2-SHIP2. A, COS-7 cells were transiently transfected with Xpress-tagged ΔSH2-SHIP2 alone or with FLAG-tagged ubiquitin, lysed, and immunoprecipitated overnight with Xpress or FLAG antibodies, respectively. Immunoblotting (IB) with anti-Xpress are shown in the inset. A fraction of the immunoprecipitation (50 and 25 μl) was used to assay phosphatase activity. The results are representative of four different experiments. In each experiments data are means of triplicates ± S.E. B, COS-7 cells were transiently transfected with His-tagged ΔSH2-SHIP2. SHIP2 in each fraction was probed by Western blotting with anti-Xpress antibodies. Tubulin α, lamin B1, and EGFR were used as controls to validate the fractionation. The percentage of ubiquitinated ΔSH2-SHIP2 over non-ubiquitinated SHIP2 in each fraction was estimated by quantitative Western blotting using anti-Xpress antibodies.