ROCK2 attenuates Rac activity underlying the formation of protrusions in migratory cells and the spine head of neurons. (A) Ratiometric images of FRET to CFP donor intensity in CHO.K1 cells expressing either the WT Raichu Rac FRET probe or as a positive control, the constitutively active Raichu Rac V12. (B) Quantification of ratiometric FRET intensity normalized to control WT Raichu Rac values; gray bars, untreated Raichu Rac V12. n ≥ 30 cells for all conditions. (C) Western blot and analysis of active Rac pulled down with PAK-PBD beads in CHO.K1 lysates, and Rac in the total lysate (bottom) similarly shows increased Rac activation in the ROCK2 knockdown cells, which is restored to control levels with coexpression of RLC-T18A, S19D (RLC-AD); n = 6 control [Ctrl], 5 ROCK1 shRNA, 6 ROCK2 shRNA, and 3 ROCK2 shRNA + RLC-AD. (D) Ratiometric images of FRET to CFP donor intensity in postsynaptic dendrites and spines of DIV 23 rat hippocampal neurons expressing either the WT Raichu Rac FRET probe or as a positive control, the constitutively active Raichu Rac V12. (E) Quantification of ratiometric FRET intensity normalized to control WT Raichu Rac values; black bars, WT Raichu Rac; white bars, Raichu Rac V12. ROCK2 knockdown increases postsynaptic Rac FRET similar to CHO.K1 cells. n (WT Raichu Rac) = 15 control and 10 ROCK2 shRNA neurons; n (Raichu Rac V12) = 12 control and 9 ROCK2 shRNA neurons. Error bars indicate SEM. MM, molecular mass.