The linker region of ANI-1 facilitates the binding of the RBD to active RHO-1 . (A) Domain organization of ANI-1 with predicted MBD, ABD, linker region, RBD, C2, and PH domain. The various GST-tagged ANI-1 C-terminal fragments used in the pull-down assay are shown. RBM - RHO-1 binding mutant (A789D, E807K). (B) His-tagged constitutive active RHO-1 (RHO-1^Q63L) or wild type RHO-1 (RHO-1^WT) were incubated with GST-tagged ANI-1^C-term, the ANI-1^C-term-RBM, or GST alone. Coomassie-stained gel of the pull-down assay shows the GST-tagged proteins and the immunoblot against the His-tag reveals the bound RHO-1 proteins (left). The mean percentages of bound RHO-1^WT or RHO-1^Q63L to the different GST-tagged fragments are shown. The amount of RHO-1^Q63L pulled down with GST-tagged ANI-1^C-term was set to 100% (right). (C) RHO-1^Q63L was incubated with GST-tagged ANI-1^C-term, the ANI-1^Link+C-term, ANI-1^Linker, or GST alone. Coomassie-stained gel of the pull-down assay shows the GST-tagged proteins and the immunoblot against the His-tag reveals the bound RHO-1^Q63L protein (left). The mean percentages of bound RHO-1^Q63L to the different GST-tagged fragments are shown. The amount of RHO-1^Q63L pulled down with GST-tagged ANI-1^C-term was set to 100% (right). For all, P values were calculated using the two-tailed Student’s t test or Mann–Whitney U test and are **P < 0.01 and ***P < 0.001, error bars are SEM, and n = number of experiments. Source data are available for this figure: SourceData F2.