The Fer-binding region in p120ctn overlaps with a RhoA-binding site, but the synaptic diffusion phenotype is independent of Rho regulation. (A and B) In vitro binding assay to map RhoA and p120ctn interaction sites. Recombinant GST-p120ctn peptides corresponding to the indicated aa were incubated with GDP-bound recombinant RhoA. (A) Representative images of RhoA-GDP bound to GST-p120ctn. The amounts of RhoA-GDP bound to GST-p120ctn were quantified in B. (C and D) Visualization of synaptophysin-GFP (Syn-GFP) at 14 DIV in neurons transfected at 10 DIV with synaptophysin-GFP together with the indicated constructs. Neurons were analyzed for the synaptophysin-GFP coverage (micrometers of GFP per 10 μm of axon length normalized to control). n = 16 (vector), 16 (Δ131–156), 18 (RhoDN), 17 (Δ131–156 + RhoDN), and 9 (RhoCA). Δ131–156, 131–156 aa-deleted p120ctn. RhoDN, dominant-negative mutant (T19N) RhoA; RhoCA, constitutively active mutant (G14V) RhoA. **, P < 0.01. Error bars represent SEM. Bar, 5 μm.