Fig. 4: Mutation in ELMO1 reduces its lipid-binding ability and decreases GEF activity of DOCK5.
From: Conformational alteration of DOCK5•ELMO1 signalosome on lipid membrane
a Lipid–protein interaction assay for ELMO1CTD. Proteins were detected using an anti-GST antibody. b Coprecipitation assay using small unilamellar vesicles (SUVs). ELMO1CTD that coprecipitated with SUVs via centrifugation at 100,000 × g were detected using SDS–PAGE followed by Coomassie Brilliant Blue (CBB) staining. c Densitometric analysis of the coprecipitated ELMO1CTD shown in (b). Data are presented as the mean ± SD (n = 9; unpaired two-sided Student’s t test, ***p < 0.005, **p < 0.01, *p < 0.05). d GEF assay using RhoGDI•Rac1 to evaluate the GEF activity of DOCK5•ELMO1CTD in the absence and presence of SUVs. Graph shows the average of three experiments, with error bars representing standard deviation (SD). e Assessment of GEF activity of DOCK5•ELMO1CTD mutants. GEF activity was assessed by measuring the exchange of GDP for Bodipy-GTP on Rac1 in the presence of SUVs composed of 78PC/20PA/2PIP3 (mol%). Data are presented as the mean ± SD (n = 3; unpaired two-sided Student’s t test, ***p < 0.005).
