Fig. 4. Mutations in the PH domain differentially abolish ARHGEF3 binding of PI(4,5)P₂ and PI(3,5)P₂.

a Ribbon representation of the structure of ARHGEF3 PH domain (PDB ID: 2Z0Q;²² image created with Pymol v2.5), with residues predicted to interact with PIP highlighted in magenta and the sidechains of those residues shown in the lower panel. b HEK293 cells were transiently transfected with EGFP-ARHGEF3 containing various mutations at the residues highlighted in (a), and the lysates were subjected to SiMPull assays against vesicles containing PI(4,5)P₂ or PI(3,5)P₂. Numbers of fluorescent molecules per image area are shown in the graphs. Data are presented as mean ± SEM. N ≥ 10 images for EGFP; N ≥ 6 images for DiD. Source data are provided as a Source Data file, which lists the exact N number for each data point. Each assay was performed with at least three independent transfections with similar results.