Fig. 10. JIP inhibits dimerization of MLK3 and V12Cdc42Hs promotes MLK3 dimerization in the presence of JIP. (A) COS 7 cells were co-transfected with Flag-MLK3 (0.1 µg), HA-MLK3 (0.1 µg) and increasing quantities of Myc-JIP plasmids as indicated. Flag-MLK3 complexes were immunoprecipitated from cell lysates using anti-Flag antibody, separated by SDS–PAGE and immunoblotted with anti-HA antibody. Corresponding cell lysates were immunoblotted to evaluate the relative expression of Myc-JIP, HA-MLK3 and Flag-MLK3 proteins. (B) COS 7 cells were transfected with Flag-MLK3 (0.05 µg), HA-MLK3 (0.05 µg), Myc-JIP (1.0 µg) and either V12Rac1, V12Cdc42Hs or L63RhoA plasmids as indicated. Where indicated, 24 h after transfection, cells were treated for 3 h with okadaic acid (400 nM). As above, immunoprecipitation and immunoblotting were used to assess the association of HA-MLK3 and Flag-MLK3 in the presence of JIP. Experiments were repeated three times with similar results.