Fig. 8. ELMO1^NTD is required for RAC1 signalling mediated by DOCK2.

a Schematic of full length ELMO1 and ELMO1^ΔNTD domains. b Expression of ELMO1^ΔNTD leads to less RAC1 activation when compared with ELMO1^WT expression. 293 T cells transfected with the indicated plasmids were subjected to a RAC activation assay (GST-PAK1 PBD pulldown). RAC activation levels were detected by western blotting and quantified using ImageJ. c DOCK2^WT binds less nucleotide-free RAC1 upon expression of ELMO1Δ^NTD. Lysates of 293 T cells expressing the indicated plasmids were used for GST-Rac1^G15A pulldown. Levels of Flag-DOCK2 bound to RAC1 were detected by western blotting and quantified by ImageJ. The experiments in (b) and (c) were performed four times. d, e Boyden migration (d) and invasion (e) assay performed with HeLa cells transfected with the indicated plasmids. Number of cells migrated were counted via DAPI staining of the membrane. (n = 3, 10 fields used at 10X per condition per experiment). f Quantifications of transfected HeLa cells displacement in a wound healing assay. Experiments were performed four times, imaging four fields using a 10X objective per condition. g Average speed of transfected HeLa cells time-lapse imaged for 6 h. Average speed was quantified using manual tracking on ImageJ. Experiments were performed four times, imaging four fields using a 10X objective per condition. Data were analysed from three experiments and expressed as mean ± s.e.m. Two-tailed unpaired student’s t-test was used (d, e, f, g) and Mann–Whitney test (b, c). (*p = 0.0286 (b, c); **p = 0.0014 (d); ***p = 0.000089 (e); *p = 0.0216 (f); **p = 0.0058 (g)). Source data are provided as a Source Data file.