spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.



Fig. 1. Identification of XPIASy as a Smad2-interacting protein. (A) An alignment of human and Xenopus PIASy (GenBank AF077952 and AF397163). Shaded amino acids are conserved residues (75% identical). (B) Phylogenetic tree of human PIAS family members and XPIASy. (C) The C-terminal region of XSmad2 binds to XPIASy in a yeast two-hybrid assay. L40 cells were transformed with pLexA-Smad2 (amino acids 180 to 432) or pLexA-Ras(G12V) and with pACTII-XPIASy, pACTII-HK-Swift (Shimizu et al., 2001), pVP-Raf or a vector, pACTII-HK. The interaction was tested by growth on SD-Trp-Leu-His plates supplemented with 10 mM 3-AT for 3 days. (D) Full-length of XSmad2 interacts with full-length XPIASy in immunoprecipitation assay. The mRNA of Flag-tagged XPIASy was injected alone (lane 3) or with XSmad2 (lanes 1,2). (E) XPIASy interacts weakly with XSmad4{alpha} or XSmad4ß but not with XSmad1. The mRNA of Flag-tagged XPIASy was injected with the indicated Myc-tagged Xenopus Smad members, and interaction was analyzed by immunoprecipitation using anti-Flag antibody. (F) Mouse PIASy interacts strongly with XSmad2 but weakly with XSmad4{alpha} or XSmad4ß. The mRNA of T7-tagged mouse PIASy was injected with the indicated Myc-tagged Xenopus Smad members, and interaction was analyzed by immunoprecipitation using the anti-T7 antibody. (G) Structures of XPIASy deletion constructs. (H) Immunoprecipitation of XPIASy deletion constructs with full-length of XSmad2. (Upper panel) A western blot of immunoprecipitated samples. The mRNA of Flag-tagged XPIASy construct was injected with Myc-tagged XSmad2 in Xenopus embryos. After immunoprecipitation against Myc, precipitated proteins were analyzed by anti-Flag staining. (Lower two panels) Before immunoprecipitation, expressed proteins were confirmed by Flag and Myc staining. (I) The N-terminal region of XPIASy interacts with the C-terminal region of XSmad2. A yeast two-hybrid assay was performed supplemented with 5 mM 3-AT, using pLexA-Smad2 (amino acids 180 to 432) and XPIASy deletion constructs subcloned into pACTII. (J) Immunoprecipitation of RING domain deleted construct, P{Delta}R. The indicated constructs were injected into Xenopus embryos. After immunoprecipitation against Myc, western blotting was performed with anti-Flag antibody.





Right arrow Return to article