FIGURE 3:

In vivo association of CPY*-GFP with Ire1. (A) The ire1Δ strain KMY1015 carrying both HA-tagged Ire1 plasmid pRS426-IRE1-HA and pRS313-TEF1pr-CPY-GFP or pRS313-TEF1pr-CPY*-GFP (or empty vector pRS313; Vector) was incubated with protein cross-linker DSP before cell lysis and anti-GFP IP. Subsequently, the lysate and the anti-GFP IP samples were analyzed by anti-HA or anti-GFP Western blotting. (B) The ire1Δ strain transformed with both pRS426-IRE1-HA (or empty vector pRS426 for lanes 1 and 7) and a GAL1 promoter-inducible CPY-GFP or CPY*-GFP plasmid, pRS313-GAL1pr-CPY-GFP or pRS313-GAL1pr-CPY*-GFP, was cultured in galactose-containing medium (see Materials and Methods for detail). After incubation with DSP, cells were lysed and analyzed by anti-HA IP, followed by anti-HA or anti-GFP Western blotting. In lanes 2, 3, and 4 and 8, 9, and 10, samples from three independent clones were analyzed. Cells for lane 6 carried an empty vector pRS313 instead of the CPY-GFP or CPY*-GFP plasmid. A molecular mass marker (M) was loaded in lane 5.