Hook1 interacts with the cytoplasmic tail of CD147 and CD98 through its carboxyl-terminal region. (A) Y2H analysis of the interaction between the cytoplasmic sequence of CD147 and the carboxyl-terminal sequence of Hook1. Yeast cells coexpressing CD147 carboxyl-terminal tail or the CD147 carboxyl-terminal acidic-cluster mutant (prey) and the carboxyl-terminal sequence of Hook1 or the pGBKT7 empty vector (bait) were grown on high-stringency plates. Growth on the –Leu/–TRP plate confirmed the expression of bait and prey plasmids (see Materials and methods for details about the Y2H screen). (B) Schematic representation of the domain organization of Hook1 (the amino terminus is shown in yellow; aa 1–168, coiled-coil region in blue; carboxyl terminus in pink, aa 659–728). Y2H clone encoding for amino acids 486–728 of Hook1. (C) BG-biotin–labeled SNAP-CD147 and CD98-SNAP were immunoprecipitated from lysates of cells coexpressing the Hook1 Y2H clone and separated on SDS–PAGE and immunoblotted as described in Materials and methods. The Hook1 Y2H clone was detected with rabbit anti-Hook1 and visualized with goat anti–rabbit 800 (bottom). The biotin-labeled SNAP-CD147 and CD98-SNAP were detected with NeutrAvidin DyLight-680 (top). For post-immunoprecipitation lysate (lanes 7–12), 1/10 of total protein was loaded.