Par1b phosphorylation inhibits the recruitment of WAVE2 and other IRSp53-binding proteins to its C-terminal, SH3-containing domain. (A and B) WAVE2 co-IPs with IRSp53 in 293 cells expressing WAVE2-GFP, Myc-tagged IRSp53 constructs (WT, A4, or IRSp53 Δ317–521; ΔCT), and Par1b where indicated. 1/10 of the lysate was analyzed for WAVE and Par1b expression. (B) Data are from three experiments with and without recombinant Par1b and three experiments without recombinant Par1b only. Error bars indicate SEM. (C) Cdc42Q61L co-IPs with IRSp53-Myc constructs from 293 cells. Arrows indicate Cdc42, and asterisks indicate IgG H and L chains. Data are representative of three experiments. (D–F) GST-IRSp53CT was subjected to Par1b phosphorylation, immobilized on glutathione and incubated with 14-3-3τ as indicated. (D) Experimental schematic is shown. (E) Binding of proteins from [³⁵S]Met/Cys-labeled 293 lysates to immobilized IRSp53CT was analyzed by Coomassie and autoradiography. Arrows indicate specific bands that are reduced in the Par1b sample. Asterisk indicates cellular 14-3-3, with one out of two identical results represented. (F) 14-3-3 binding to IRSp53CT was analyzed by IB, and IRSp53-CT levels by Ponceau red stain are shown.