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. 2007 Oct 8;179(1):75–86. doi: 10.1083/jcb.200704166

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Copyright © 2007, The Rockefeller University Press

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Figure 2. — Cluster formation of Ire1. Excepting E, Ire1-HA variants were expressed from wild type (WT) or mutant versions of pRS315-IRE1-HA (D; centromeric plasmid) or pRS423-IRE1-HA (A–C and E–I; 2-μm plasmid) in yeast strain KMY1015 (ire1Δ; D), YKY1004 (ire1Δ hac1Δ; G), or KMY1516 (ire1Δ; A–C, E and F, and H and I) and detected by anti-HA immunofluorescent staining. For vector control not carrying the Ire1-HA gene, cells carrying pRS423 (A) or pRS315 (D) were used. (E) An Ire1-HA gene knockin strain YKY2005 was examined. Where indicated, Tun (2 μg/ml final concentration) or DTT (10 mM final concentration) was added into cultures 1 h before harvest. (F) Cells treated with 10 mM DTT for 30 min were washed with medium and further incubated for the indicated times. Cells were observed by a conventional fluorescent microscope, and exposure times for image acquisition were 2 s for D and E and 1 s for other panels.