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Figure S3. Live cell MT-binding assay. (A) Wild-type KHC becomes locked on MTs upon AMPPNP exposure, but a mutant KHC that cannot bind to MTs does not. COS cells expressing KHC-mCit (left) or a mutant KHC in which the MT-binding loop has been mutated (Δloop12; right) were untreated (top) or permeabilized with streptolysin O (SLO) and exposed to AMPPNP for 10 min (bottom). The cells were then fixed and stained with a mAb to α-tubulin. Bar, 20 µm. (B) Neither JIP1 nor FEZ1 alone can activate KHC + KLC. COS cells transfected with the indicated plasmids were exposed to AMPPNP for 10 min and were fixed and stained. Fluorescence images of KHC-mCit (left), KLC-mCFP (middle), and immunostaining of JIP1 or FEZ1 (right) are shown. Bar, 25 µm. (C) Model for the activation of Kinesin-1. JIP1 binding to the KLC subunit and FEZ1 binding to the KHC tail are both required to relieve autoinhibition of Kinesin-1.