Fig. 2: WWC proteins interact directly with and stabilize Motins.
From: WWC1/2 regulate spinogenesis and cognition in mice by stabilizing AMOT
A–C AMOTp130 can interact with WWC1 (A), WWC2 (B), and WWC3 (C). HEK293A cells were co-transfected with the indicated plasmids. Cell lysates were immunoprecipitated (IP) with anti-FLAG beads, and then examined by immunoblotting using the indicated antibodies. D Both WW domains in WWC1 mediate interaction with AMOTp130. HA-AMOTp130, WW1mut (W34A/P37A), WW2mut (P84A), WW1/2mut (W34A/P37A/P84A) and WT WWC1 (FLAG-tagged) were expressed in HEK293A cells and subjected to co-immunoprecipitation assays. E PPxY motifs in AMOTp130 mediate interaction with WWC1. HA-WWC1, LPTY106LATA, PPEY239PAEA, PPEY284PAEA, PPEY239/284PAEA and WT AMOTp130 (FLAG-tagged) were expressed in HEK293A cells and used for co-immunoprecipitation assay. F, G WW domains in WWC1 are required for the stabilization of AMOTp130. Whole-cell lysates from vector, WT WWC1, and WWmut WWC1 overexpressed WWC1/2/3 tKO HEK293A cells treated with cycloheximide (CHX; 100 mg/mL for 2–10 h) were collected and subjected to immunoblotting (F). Quantification is shown in G. Data are shown as the mean ± SD of three independent experiments. H, I A patient-derived WWC1 mutant (W88C) is unable to stabilize AMOTp130. WT or W88C mutant WWC1 was overexpressed in WWC1/2/3 tKO HEK293A cells and treated with cycloheximide (CHX; 100 mg/mL for 2–10 h). Whole-cell lysates were then collected and subjected to immunoblotting (H). Quantification is shown in I. Data are shown as the mean ± SD of three independent experiments.
