Figure 5.

POSH and Shroom3 coassociate. (A) A yeast two-hybrid screen identified Shroom3 as an interacting partner for POSH. Alignment showing the domain structure of Shroom3L (aa 1-1986) and Shroom3S (aa 177-1986), the location of the Shroom3 two-hybrid isolates, and the number of times each class of isolate was recovered from the screen. PDZ, PSD-95/Dgl/ZO-1 (aa 1-106). ASD1/2, Apx/Shroom domain 1/2 (aa 927-1028 and aa 1659–1983, respectively). ABD, F-actin binding domain (aa 754-952). EVH1 BS, Enabled/vasodilator-stimulated phosphoprotein homology 1 domain binding site (aa 1533-1539). PDZ BS, PDZ domain binding site (aa 1983-1986). (B) Direct interaction between POSH and Shroom3. GST-POSH SH3-3 or GST and His epitope-tagged Shroom3 POSH binding domain (PBD) fusion proteins were purified from bacteria, allowed to bind in vitro, then His-Shroom3 was immunoprecipitated (IP). His-Shroom3 and coassociated GST proteins were detected by Western blot analysis (WB) with antibodies directed against the epitope tags after SDS-PAGE. (C) Endogenous POSH and Shroom3 coassociate. Shroom3 was immunoprecipitated from E16.5 mouse embryonic brain extracts (left) or IMR32 human neuroblastoma cells (right). An irrelevant goat polyclonal antibody was used as a specificity control (Ctrl). After SDS-PAGE, Western blot analysis was used to detect Shroom3 and coassociated POSH.