Fig. 2: A SCRIB-AFDN complex is mediated by the FHA ___domain of AFDN and the PDZ1 ___domain of SCRIB.

a Localization of AFDN and SCRIB in HeLa cells. EGFP-AFDN and immunostained FLAG-SCRIB were imaged by confocal microscopy. Scale bar represents 10 µm. b Endogenous SCRIB and AFDN co-localize at cell contacts in MCF7 cells. Antibody specificity to endogenous AFDN/SCRIB is verified in Fig. 6a. Projections of z-stacks are at right and top of the merged images, with position shown by dashed yellow line. Scale bar represents 10 µm. c Full-length AFDN and SCRIB co-immunoprecipitate (IP). EGFP-tagged SCRIB and FLAG-tagged AFDN were co-expressed in HEK 293T cells. IP with anti-FLAG followed by Western blotting with anti-GFP revealed the complex. Vector alone was a control. d Endogenous AFDN and SCRIB co-precipitate from MDCKII cells. Following AFDN IP, an anti-SCRIB blot confirmed the complex. No AFDN or SCRIB were observed in an anti-Myc IP (control). e Seven fragments comprising the whole of AFDN were generated to map the interaction, generally encompassing the AFDN modular domains. f SCRIB is precipitated by the FHA ___domain of AFDN. GST-fusions of the 7 AFDN fragments were mixed with lysates expressing KRAS, activated KRAS-G12V, SCRIB, or the C/N-terminal regions of SCRIB. Full-length recombinant and endogenous SCRIB and its N-terminal half associated with AFDN FHA. Activated KRAS bound the RA domains. g Fragments of SCRIB used for mapping its interaction with AFDN. h AFDN was precipitated by the PDZ1 ___domain of SCRIB. GST-fusions of the SCRIB LRR or PDZ1 domains were mixed with lysates expressing AFDN. Both isoforms and endogenous AFDN were bound by PDZ1. i AFDN-SCRIB complex is dependent on the FHA ___domain of AFDN. Full-length AFDN or an FHA deletion mutant were co-transfected with SCRIB. Following anti-GFP IP, an anti-FLAG blot revealed SCRIB association is disrupted by AFDNΔFHA. j Complex between AFDN and SCRIB is dependent on the PDZ1 of SCRIB. Full-length or a PDZ1 deletion mutant of SCRIB were co-transfected with AFDN. Following anti-GFP IP, an anti-FLAG blot revealed SCRIBΔPDZ1 does not precipitate AFDN. All source data are provided in the Source Data files.