Figure 2

DCX C-terminal deletion increases DCX association with MTs and induces different MT bundling patterns. (A) GFP-tagged DCX full-length (wild-type, WT) and a DCX C-terminal truncation mutant lacking residues 276–366 (DCX ΔC) were created to test the impact of the DCX C-terminus on the association of DCX with MTs. (B) Following transfection into COS-1 cells, equivalent expression of the GFP-DCX constructs was detected by immunoblotting for GFP (upper panel), with equivalent protein loading detected by immunoblotting for total α-tubulin (lower panel). Blots for both GFP and α-tubulin were cropped. (C) GFP-DCX constructs were visualised as green by confocal laser scanning microscopy (left panels), and the impact on endogenous tubulin organisation was evaluated by staining with the live imaging dye SIR-tubulin (red, middle panels); merge images indicate the areas of colocalization (yellow) of GFP and SIR-tubulin. Scale bars represent 10 µm. (D) The impact on endogenous tubulin organisation was evaluated by the Pearson correlation coefficient. (E) Cells expressing GFP (control vector), GFP-DCX WT and GFP-DCX ΔC were incubated with GFP-trap beads overnight. The GFP-trap beads bound to GFP-tagged constructs were separated from the supernatant by centrifugation. (i) The pellet fraction contains GFP-tagged constructs bound to tubulin (DCX-bound tubulin) and (ii) the supernatant fraction contains soluble tubulin (unbound tubulin). Immunoblotting was performed to detect endogenous α-tubulin (upper panel) or GFP (lower panel). Blots for α-tubulin were cropped. (F) Co-precipitation of MTs with GFP-DCX proteins was estimated from quantitating the tubulin/GFP ratio in each pellet fraction containing DCX-bound tubulin. Error bars represent the standard error of the means and asterisks indicate values calculated to be statistically significantly different (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 n = three independent experiments).