Figure 3
Binding assay and co-immunoprecipitation assay to show the interaction between Ha-Ntf2 and Ha-Ran in vitro or in vivo. A, B: SDS-PAGE to show the direct binding assay between the rHa-Ntf2 and rHa-Ran in vitro. A, rHa-Ntf2 binds on His-rHa-Ran affiliated on Ni2+-NTA column. B, rHa-Ran binds on His-rHa-Ntf2 affiliated on Ni2+-NTA column. rHa-Ntf2 or rHa-Ran: removed His-tag by thrombin; dHis-rHa-ntf2: the degradation of His-rHa-ntf2 in the process of binding. C, D: immunoblotting to examine the interaction of Ha-Ntf2 and Ha-Ran produced by co-immunoprecipitation. C, Ha-Ntf2 detection from the co-immunoprecipitation (co-ippt) produced by anti-Ha-Ran antibody. Lane 1, protein extracts from the whole larvae of 6th-96 h; lane 2, sample from the last wash of the co-ippt; lane 3, eluted proteins from well-washed co-ippt produced by anti-Ha-Ran antibody. D, Ha-Ran detection from the co-ippt produced by anti-Ha-Ntf2 antibody. Lanes 1 and 2 are the same as in C; lane 3, eluted proteins from well-washed co-ippt produced by anti-Ha-Ntf2 antibody. M, protein marker.