In vitro binding assay. (A) Each column of micrographs shows the GDP-MTs (labeled with Alexa Fluor 488), GMPCPP-MTs (labeled with Alexa Fluor 647), and kinesin motor domain (KIF5C, labeled with Alexa Fluor 594). Numbers in the kinesin column indicate the concentration of the kinesin motor domain (nM). Bar, 10 µm. (B) Histograms to show the fluorescence intensity of Alexa Fluor 594 channel (kinesin motor domain) bound to GDP-MTs (recognized by Alexa Fluor 488 channel signal, red) and to GMPCPP-MTs (recognized by AlexaFluor 647 channel signal, blue). The numbers in each panel show the final concentration of kinesin motor domain in the assay mixture. For each kinesin concentrations, 5–7 view fields were analyzed, which contained ∼100 GDP-MTs and 150–300 GMPCPP-MTs. (C) The ratio of bound kinesin between GMPCPP-MTs and GDP-MTs, plotted against kinesin concentration. The ratio was fitted with simple binding kinetics: where and are the dissociation constants for GMPCPP-MTs and GDP-MTs, respectively. The mean and SEM of the same dataset as B and the best fit curve (and) are shown. The data shown are from two separate replicates of the experiment, which are representative of five replicates of the experiment.