Fig. 4

NDR2 interacts and phosphorylates GEF-H1 in HBEC. HBEC-3 or H1299 cells were transfected with siNeg and/or siRASSF1A, siNDR1, siNDR2, siGEF-H1, pcDNA3-NDR1, pcDNA3-NDR2 or pcB6-GEF-H1. a GST-RBD pull-down assay in H1299 cells. b Ser885 phosphorylation from GEF-H1 assayed by western blot following λ-phosphatase pre-treatment of the total protein extract or not and normalized with total GEF-H1 expression in HBEC-3 cells. c GST-NDR1 or -NDR2 pull-down assay using siRNA & GEF-H1 plasmid as controls. NDR2 phosphorylation on Ser265-GEFGH1 link with phosphorylation on Ser-885-GEF-H1A was assayed. NDR2 activity on HBEC-3 cells extracts previously transfected with pcB6-GEF-H1 wild type, mutated on Ser265 (S265A), or on Ser885 (S885A) was assayed by quantifying ser885-GEF-H1 phosphorylation status by western blot following normalization by total GEF-H1 expression. Error bars indicate the SEM (n ≥ 3). *P < 0.05, **P < 0.01 and ***P < 0.001, using an ANOVA test followed by Dunnett’s test