Figure 7.
Yeast two-hybrid and affinity chromatography assays identify a direct binding interaction between DIC and NF-M. A fragment from the rod domain of NF-M spanning amino acid residues 122-428, identified as a DIC-binding protein in a yeast two-hybrid screen, was specifically bound to a DIC affinity matrix. A similar fragment from NF-L as well as full-length NF-M and NF-L polypeptides, did not bind significantly to DIC in either the yeast two-hybrid or affinity chromatography assays. For affinity chromatography, in vitro-translated proteins (L) were loaded in parallel onto DIC or BSA affinity columns, and the flowthrough (F), wash (W), and elution (E) fractions were analyzed by SDS-PAGE and autoradiography. Interactions were also assayed pairwise in the yeast two-hybrid assay and scored vs. positive (p150Glued) and negative (vector only) controls.