Fig. 3

PRICKLE1 is associated to the Rho-GEF ECT2 and controls its activity. a Immunopurification of GFP-PRICKLE1 from MDA-MB-231 cell lysate using GFP nanobodies coupled to sepharose beads allows the identification of ECT2 associated to PRICKLE1. b Immunofluorescence of MDA-MB-231 cells stably expressing GFP-PRICKLE1 shows that ECT2 (endogenous) is colocalised with PRICKLE1 and enriched in actin structures within the lamellipodia. c Mapping of the PRICKLE1 domain needed for interaction with ECT2. HEK293T cells were co-transfected with the indicated forms of PRICKLE1 (see on the left for topology details) and mCherry-ECT2. After FLAG immunopurification, presence of ECT2 is detected using anti-mCherry antibody. d Downregulation of PRICKLE1 expression using siRNA targeting PRICKLE1 shows an increase of Rac activity in MDA-MB-231 cells. e PRICKLE1 modulates ECT2 activity. Using HEK293T cells, we expressed or co-expressed ECT2 with full length or a deleted version of PRICKLE1 lacking its domain of interaction with ECT2. Overexpression of ECT2 leads to an increase in Rac activity which was inhibited when PRICKLE1 is co-expressed. Co-expression of a mutant form of PRICKLE1 did not modify the gain of function observed by ECT2 overexpression