Fig. 3.

TIPE2 binding to Rac requires the C-terminal CAAX motif, and TIPE2 inhibits Rac membrane translocation. (A) Schematic diagram of the Rac1 protein. (B–D) Lysates of 293T cells transiently transfected with HA-Rac1 constructs and Flag-TIPE2 construct were immunoprecipitated with anti-Flag and subjected to Western blotting. (E) 293T cells were transiently transfected with increasing amounts of TIPE2 plasmids for 8 h. Cell lysates were separated into membrane and cytosolic fractions. Equal amounts of cytosolic and membrane materials (3 μg) were separated by SDS/PAGE and immunoblotted for integrin β1, β-actin, and Rac. (F) Lysates of 293T cells transiently transfected with or without HA-Rac1 construct and Flag-TIPE2 constructs were immunoprecipitated and subjected to Western blotting. (G) 293T cells were transiently transfected with or without the indicated TIPE2 plasmids. Equal amounts of cytoplasmic and membrane protein fractionations were subjected to Western blotting. (H) WT BMDMs were treated with LPS for up to 16 h. Cytosolic and membrane protein fractionations were subjected to Western blotting. The experiments were repeated at least three times with similar results.