Fig. 2. Direct in vitro interaction of ß-dystrobrevin and neuronal kinesin heavy chain. (A) Co-immunoprecipitation: cMyc-ß-dystrobrevin and HA-kinesin (Kif5A clone 18) were translated in vitro in the presence of [³⁵S]methionine and [³⁵S]cysteine, and mixed together. The mixtures were immunoprecipitated with polyclonal anti-HA, and the immunoprecipitated proteins visualized by autoradiography after SDS-PAGE. Non-specific IgG were used as negative control. ³⁵S-labeled cMyc-ß-dystroglycan (amino acids 774-895) did not co-immunoprecipitate with kinesin, showing the specificity of the ß-dystrobrevin interaction with kinesin. (B) Pull-down assay: purified GST-ß-dystrobrevin (GST-ß-DB) or GST-protein (GST), pre-bound to glutathione-Sepharose beads, was incubated with in vitro translated [³⁵S]kinesin (Kif5A clone 18) or [³⁵S]Dp71 (Dp71_285-622, used as a positive control). After extensive washing, bound radioactive proteins were separated by SDS-PAGE and detected by autoradiography. The extra band under [³⁵S]kinesin in the first lane is a product present after the in vitro transcription and translation.