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. 2008 Aug 11;182(3):531–542. doi: 10.1083/jcb.200711151

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© 2008 Ju et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 2. — KDEL-tagged soluble form of Cosmc functions as wtCosmc and localizes in ER. (A–C) Function of KDEL-tagged soluble form of Cosmc. 293T cells were transiently transfected with plasmids encoding T-synthase–HPC4, HPC4-sCosmc, HPC4-sCosmc-KDEL, and wtCosmc. Cell extracts were prepared and T-synthase activity was measured. Bar 1 represents a single value, bar 5 represents a mean of duplicate values with error bar shown, and the remaining bars represent a mean of triplicate values with error bars shown. The pink line indicates the endogenous level of T-synthase activity and the green line represents the activity after transfection of T-synthase alone (A). T-synthase and Cosmc in cell extracts and media were also analyzed by Western blot with anti-HPC4 (B and C). Black lines indicate that intervening lanes have been spliced out. (D–F) Localization of sCosmc-KDEL. CHO K1 cells cultured on chambered slides transiently transfected with HPC4-sCosmc-KDEL were immunofluorescently stained with anti-HPC4 (green; D) and rabbit anti-calnexin IgG (red; E) and merged (F). The images were collected by confocal microscopy. Bars, 8 μm.