Fig. 5. Self-association of the Ire1 luminal domain mutants. (A) KMY1015 (ire1 null mutant) cells carrying both pRS426-Ire1-Flag (2-µm plasmid for expression of Ire1-Flag; wild type (WT) or mutant) and pRS315-Ire1-HA (WT or mutant) were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes, and lysates were then used for anti-HA immunoprecipitation. The cell lysates (equivalent to 3x10⁶ cells) and the anti-HA immunoprecipitates (equivalent to 1x10⁷ cells; -HA IP) were analyzed by anti-HA (-HA) and anti-Flag (-Flag) western blotting (WB). The ratios of Ire1-Flag signal to Ire1-HA signal in -HA IP were normalized to the Tun+ WT Ire1 control, which was set at 1.0, and are indicated. (B) KMY1015 cells carrying pRS315-Ire1-HA (core or S103P core mutant) were cultured with (+) or without (–) 2 µg/ml tunicamycin (Tun) for 60 minutes, and their lysates were fractionated by 5-25% glycerol gradient centrifugation. Ire1-HA in each fraction was detected by anti-HA immunoprecipitation followed by anti-HA Western blotting. The positions of protein M_r markers, fractionated in parallel on an identical gradient, are indicated.