(A) Western blot analysis of coimmunoprecipitation assays showing co-operative formation of the JIP1 and JIP3 complex. The effect of the KLC1TPR R266S mutations on the formation of the complex is also shown. (B, C) Quantification of relative JIP1 binding (B) and JIP3 binding (C) from three independent co-immunoprecipitation experiments (normalized to the sample indicated by a black bar). Error bars show SEM. Statistical analysis was performed with the GraphPad Prism package using ordinary one-way ANOVA for multiple comparisons, *p<0.05, ***p<0.001. (D, E, F) Binding of GST-JIP3LZ2 to His6-KLC1extTPR (D), His6-KLC1extTPR-JIP1C-term (E), His6-KLC1TPR-JIP1C-term (F) measured by ITC. It shows that JIP3LZ2 affinity for the TPR domain is not affected by either the LFP-acidic region or JIP1C-term loading. All experiments were carried out in 50 mM HEPES, 500 mM NaCl, 5 mM 2-mercaptoethanol at T = 20°C. In (D) the volume of the first six injections following the initial sacrificial one was 1.0 μl and the rest were 2.0 μl. In (E) and (F) all working injections were 1.5 μl. All injections were performed at 150 s time intervals.