Fig. 6
DEF6 mutations affect RAB11 interactions. a Representative images of endogenous CTLA-4, and RAB11 and DEF6 in TCR-stimulated healthy control (HD) PBMCs showing RAB11-CTLA-4 co-localization. b Line scans of images in (a) reveal high overlap of RAB11 and CTLA-4 signal in activated HD-PBMCs. Scale bar – 5 µm. c Representative images of endogenous CTLA-4, RAB11 and DEF6 in activated PBMCs of P1 reveal loss of RAB11-CTLA-4 co-localization in P1. d Line scans show reduced overlap of RAB11 and CTLA-4 signal in P1. Scale bar – 5 µm. (for a-d, representative images of 30–40 analyzed cells; cells were considered T cells through expression of CTLA-4 after TCR cross-linking). Quantification as in Fig. S5a. e MYC-tagged wildtype DEF6 co-immunoprecipitates with GFP-RAB11 from transfected HEK293T cells, revealing a hitherto unrecognized interaction of the GEF protein DEF6 with the small GTPase RAB11. Presence of mutation E331K abrogated this interaction. Samples were balanced on immunoprecipitated GFP-RAB11 fractions and blotted for interacting DEF6. Representative of three independent experiments. f Endogenous DEF6 co-immunoprecipitates from Jurkat lysates with overexpressed RAB11-Strep-HA, compared to GFP-Strep-HA control. Samples were balanced on immunoprecipitated HA-tag fractions. Western blots (e, f) were cropped for visualization. g, h Overexpressing inactive RAB11S25N in Jurkat-mCherry-CTLA-4 cells mimics DEF6-deficient defects in CTLA-4 cycling. Cells were electroporated with inactive RAB11S25N, constitutively active RAB11Q70L, wildtype RAB11 or empty vector (EV), stimulated with OKT3 and analyzed for cycling CTLA-4 after 10 min and 30 min, respectively. Cycling was reduced in RAB11S25N expressing cells (blue, blue numerical insert, g), while RAB11Q70L enhanced cycling at 30 min (h). Data are overlaid with mean ± SD. Representative of two individual experiments. Source data of Fig. 6 including uncropped immunoblots are provided as a Supplementary Source Data file
