Fig. 5. Dot-blot overlay assays of purified recombinant polymerized intermediate filament vimentin (B) and desmin (C) with radiolabeled fragments of the DP tail. ³⁵S-radiolabeled c-myc-tagged recombinant forms of DP, DP-BC^S2849G, DP-BC51, DP-BL, DP-C^S2849G, DP-C51 and DP-L were generated by coupled in vitro transcription/translation and analyzed by SDS-PAGE and autoradiography (A). The occasional detection of additional radiolabeled protein bands most likely reflect the presence of partially transcribed and translated products (A). Different amounts of polymerized vimentin (B) and desmin (C) were immobilized on a nitrocellulose membrane by dot blotting and incubated with radiolabeled DP-BC^S2849G, DP-BC51, DP-BL, DP-C^S2849G, DP-C51, DP-L. Protein loading was verified by amino-black staining of the spotted proteins (P). The binding efficiency of DP-BC^S2849G to both desmin and vimentin varied according to the amount of immobilized filaments. While binding of DP to desmin abruptly decreased when the amount of desmin was below 0.5'g, the association of DP with vimentin was maintained for up to 0.05'g immobilized vimentin. Furthermore, DP-BC51 and DP-BL exhibited greatly reduced binding for both desmin and vimentin compared with DP-BC^S2849G. Finally, DP-L, DP-C^S2849G, and DP-C51 did not detectably interact with either desmin or vimentin.