Figure 4. Immunoprecipitation assay to demonstrate the dimer-forming ability of core stress-sensing region in vivo.
(A) Diagram of constructs encoding full-length Ire1 or Ire1(ΔC), and its Δ145 and Δ327 mutants respectively. These proteins were C-terminally tagged with the 3× FLAG and 3× HA epitopes respectively. TM means transmembrane region (black). (B) A Δire1 strain KMY1015 was transformed with pRS315-Ire1(ΔC)-HA and pRS426-Ire1-FLAG, or their internal deletion mutants. The transformants carrying both plasmids were selected and cultured with (+) (lanes 2, 4 and 6) or without (−) (lanes 1, 3 and 5) 2 μg/ml tunicamycin (Tu) for 60 min, and subjected to anti-HA immunoprecipitation (IP) as described in the Experimental section. The cell lysates (equivalent to 3×106 cells) and immunoprecipitates (equivalent to 1×107 cells) were analysed by anti-HA (α-HA) and anti-FLAG (α-FLAG) Western blotting. Lanes 1 and 2, wild-type (WT) luminal region [Ire1(ΔC)–HA and Ire1–FLAG]; lanes 3 and 4, Δ145 mutants [Ire1(ΔC)–HA:Δ145 and Ire1–FLAG:Δ145]; lanes 5 and 6, Δ327 mutants [Ire1(ΔC)–HA:Δ327 and Ire1–FLAG:Δ327].