Fig. 4. PIASy interacts with coilin in vitro and in vivo. (A) Yeast cells transformed with PIASy prey and the indicated coilin baits were spotted onto non-selective (–LW) or selective (–LWHA) media and incubated for 3 days. Note that yeast harboring PIASy prey with coilin baits containing the first 161 or 92 amino acids showed minimal growth on the selective medium, as did yeast with PIASy prey and the C-terminal 214 aa of coilin (362-576) as bait. (B) His-T7-tagged full-length (FL, 1-576) or N-terminal fragments (1-161, 1-362) of coilin, detected by anti-coilin or anti-T7 antibodies, bind GST-PIASy but not GST alone. By contrast, a C-terminal fragment of coilin containing the last 214 aa of coilin (C214) does not interact with GST-PIASy. Ponceau S staining verified that equivalent amounts of GST proteins were used in the assay. The input lanes account for 20% of the coilin or coilin truncations used in the reactions. (C) HeLa cells were co-transfected with empty GFP vector, GFP-coilin or GFP-coilin mutants along with myc-tagged PIASy. GFP-C214 corresponds to the C-terminal 214 aa of coilin (from aa 362-576). Lysates were subjected to immunoprecipitation with anti-GFP antibodies, followed by western blotting with anti-myc antibodies (upper rows in all panels). The blots were reprobed with anti-GFP to monitor the level of the GFP tagged proteins (lower rows in all panels). The input lanes account for 2% the amount of lysate used in the immunoprecipitation reactions. (D) HeLa cells were transfected with empty GFP vector or GFP-PIASy, followed by immunoprecipitation with anti-GFP antibodies and western blotting with antibodies against coilin (top panel) or GFP (bottom panel). The input lane accounts for 2% the amount of lysate used in the immunoprecipitation reactions.