Fig. 5: DCLK1 isoforms and the effect of DCLK1-IN-1 on microtubule polymerisation and binding function.

a Domain diagrams of DCLK1 isoforms based on the Uniprot classification. b The difference in melting temperature (T_m) of DCLK1-KD, DCLK1-FL1Δ D511N and DCLK1-FL1Δ WT in the presence of DCLK1-IN-1 and DCLK1-NEG. The difference in the T_m was calculated from the melting profiles shown in Supplementary Fig. 8. c Representative fitted SPR sensorgrams for DCLK1-IN-1, FMF-03-055-1, or XMD8-85 binding to immobilised DCLK1-FL1Δ D511N, showing mean values from kinetic fitting to a 1:1 binding model (K_D, dissociation constant; k_on, on-rate; k_off, off-rate; t_1/2, dissociative half-life for the protein/inhibitor complex). Data represent an average of either four (DCLK1-IN-1) or three (FMF-03-055-1 and XMD8-85) independent experiments, each performed as duplicate titrations; errors are SEM. See also Supplementary Fig. 9. d Microtubule polymerisation and pelleting assays followed by SDS-PAGE gel analysis of pellet (P) and supernatant (S) fractions. Tubulin polymerisation was tested in the presence of DCLK1-FL1Δ D511N and DCLK1-FL1Δ WT. The microtuble destabiliser, nocodazole, was used as a negative control. DCLK1-FL1Δ D511N was incubated with DCLK1-IN-1 (20 and 40 μΜ) or nocodazole (40 μΜ) prior to the addition of tubulin. This gel is a representation of samples tested in two independent experiments. e Fluorescence-based Tubulin Polymerisation Assay in the presence of DCLK1-FL1Δ D511N. Paclitaxel (3 μΜ), a microtubule stabiliser was used as a positive control and nocodazole and tubulin buffer alone were used as negative controls. DCLK1-FL1Δ D511N was incubated with DCLK1-IN-1 (20 and 40 μΜ) or nocodazole (80 μΜ) prior to the addition of tubulin. This curve is a representation of samples tested in duplicates and in two independent experiments. Data points are shown for the first 30 min as tubulin polymerisation reached the steady state. See also Supplementary Fig.10.