Krz preferentially interacts with inactive ERK. ( A ) S2 cells were transfected with HA–Krz, HA–Krz-K336A, HA–Krz-R209E or HA–Krz-R209E-K336A together with Drosophila ERK–Flag. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. ( B ) S2 cells were transfected with Drosophila ERK–Flag alone, or together with HA–Krz-R209E or HA–Krz. Cells were treated with insulin at a final concentration of 20 μM. Samples were immunoprecipitated with anti-Flag or anti-HA beads. Immunoprecipitates were analysed by western blot with anti-HA antibody, anti-total ERK antibody and anti-dpERK antibody. ( C ) S2 cells were transfected with HA–Krz or HA–Krz-R209E together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. ( D ) S2 cells were transfected with HA–Krz and either Drosophila wild-type ERK–Flag or ERK-D334N–Flag. Lysates were immunoprecipitated with anti-HA beads and immunoblotted. ( E ) S2 cells were transfected with HA–Krz together with the indicated Drosophila ERK–Flag versions. Samples were immunoprecipitated with anti-HA beads and analysed by western blotting. IP, immunoprecipitated samples; IB, immunoblots.