Kinesin heavy chain also binds to the RH1 domain of JIP3 but does not compete for binding with dynein light intermediate chain. (A and B) Left: Coomassie Blue-stained SDS-PAGE gel of purified recombinant protein mixtures prior to the addition of glutathione agarose resin (Input). Right: Coomassie Blue-stained SDS-PAGE gel of proteins eluted from glutathione agarose resin after GST pull-down. Proteins correspond to the human homologs and to mouse kinesin heavy chain KIF5C (UniProt entry P28738). Molecular weight is indicated in kilodaltons (kD). (C and D) Elution profiles (top) and Coomassie Blue-stained SDS-PAGE gels (bottom) of purified recombinant human proteins after size exclusion chromatography on a Superdex 200 Increase 10/300 GL column. The elution profile and gel for GST::DLIC1[388-523] are shown twice in C, and the elution profile and gel for GST::KIF5C[807-956] are shown twice in D. Molecular weight is indicated in kilodaltons (kD). WT denotes wild type. (E) Left: Coomassie Blue-stained SDS-PAGE gel of purified recombinant protein mixtures prior to the addition of amylose resin (Input). Right: Coomassie Blue-stained SDS-PAGE gel of proteins eluted from amylose resin after MBP pull-down. The actual amount of DLIC1[388-523] in the pull-down reaction was fivefold higher than what is shown for the input. The KIF5C fragment was used at two concentrations that differ threefold, as indicated above the 5th and 6th lanes from the left. (F) Coomassie Blue-stained SDS-PAGE gel (top) and corresponding immunoblot (bottom) of proteins eluted from amylose resin after MBP pull-down as in E. All proteins contain a 6xHis tag (see Materials and methods) that is detected on the immunoblot. Proteins are the same as in E, but amounts in the pull-down mixture were decreased relative to those in E such that DLIC1-C helix 1 peptide could be added in 150-fold molar excess over the KIF5C fragment. Source data are available for this figure: SourceData F3.