Fig. 4: RAS GTPases complex with AFDN-SCRIB.
From: Afadin couples RAS GTPases to the polarity rheostat Scribble

a Activated KRAS is co-precipitated with AFDN and SCRIB. HEK 293T cells were co-transfected with vectors expressing FLAG-AFDN and EGFP-KRAS-G12V (constitutively active). Both endogenous SCRIB and KRAS-G12V were detected following anti-FLAG IP. FLAG vector alone was a control. b Activated KRAS and AFDN co-IP with SCRIB. HEK 293T cells were co-transfected with vectors expressing FLAG-SCRIB and EGFP-KRAS-G12V. Endogenous AFDN and KRAS-G12V were detected following anti-FLAG IP. c The RA1/2 domains of AFDN interact with multiple RAS small GTPases. EGFP-tagged GTPases with activating mutations were expressed in HEK 293T cells, and GST-tagged RBD domains from BRAF or AFDN purified from bacteria. GST-RBDs bound to glutathione beads were used to pull down (PD) GTPases, a Western blot with anti-GFP revealed interacting proteins. d Specificity of individual AFDN RA domains for RAS GTPases. GST-tagged RA1, RA2 or RA1/2 were purified and mixed with lysates expressing the indicated GTPases. An anti-GFP immunoblot following precipitation on glutathione beads revealed bound GTPases. Each blot was exposed for 30 seconds. e Activated GTPases co-precipitate with AFDN and augment interaction with SCRIB. Full-length, EGFP-tagged SCRIB or activated RAS family GTPases were co-transfected with FLAG-tagged AFDN. Following immunoprecipitation with anti-FLAG, Western blot with anti-GFP revealed activated KRAS, RAP2C or RAP1B complex with AFDN and SCRIB. f SCRIB lacking PDZ1 does not co-precipitate with AFDN and does not prevent the association between AFDN and KRAS. Full-length wild-type or ΔPDZ1 SCRIB were co-transfected with AFDN and KRAS. Association with SCRIB and KRAS was detected by Western blot following anti-FLAG immunoprecipitation of AFDN. g Quantitation of SCRIB binding to AFDN when co-expressed with activated GTPases. The amount of EGFP-SCRIB co-precipitating with AFDN in anti-FLAG Western blots was determined by densitometry (n = 4 for RAP1B/RAP2C and n = 7 for KRAS, from distinct experiments). The ratio is the amount of SCRIB detected when co-expressed with RAP1B-G12V (P = 0.018), RAP2C-G12V (P = 0.020) or KRAS-G12V (P = 0.007), versus wild-type KRAS. Line represents the median, box the interquartile range (IQR) and whiskers the min/max. **P < 0.01, *P < 0.05 as measured by paired, two-tailed t-test. All source data are provided in the Source Data files.