FIGURE 5.

Phosphorylation of PREX2 prevents binding to the membrane, PIP₃, and Gβγ. A, Western blot analysis of cytosolic and membrane fractions from starved or insulin-treated HEK293 cells expressing V5 PREX2 with or without co-expression of FLAG/HA Gβγ. B, Western blot analysis of GST PTEN pulldowns of endogenous PREX2 from cytosolic and membrane fractions of HEK293 cells. Cells were starved and then treated with insulin for indicated times. C, Western blot analysis of PIP₃ pulldowns from starved or insulin-treated HEK293 cells expressing V5 PREX2 with or without FLAG/HA Gβγ. D, Western blot analysis of endogenous PREX2 isolated from HEK293 cells with either PIP₃ beads (top) or PREX2 antibody (bottom). Cells were starved and then treated with DMSO or 500 nm GDC0941 followed by treatment with insulin. E, Western blot analysis of FLAG immunoprecipitations from HEK293 cells co-expressing FLAG/HA Gβγ and V5 PREX2 with and without 100 nm calyculin A treatment. F, Western blot analysis of pulldowns with GST PTEN, GST PP1α, GST Rac1, or PIP₃ beads from HEK293 cells expressing V5 PREX2 with or without FLAG/HA Gβγ. The cells were starved and treated with insulin or 100 nm calyculin A. U, upper band; L, lower band.