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. 2016 May 15;27(10):1650–1662. doi: 10.1091/mbc.E16-01-0033

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© 2016 Inoue and Tsai. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Grp170 is essential in degradation of the glycosylated NHK ERAD-Ls substrate. (A) WCEs derived from 293T cells transfected with the indicated plasmid were incubated with S-protein–conjugated beads and the isolated proteins subjected to SDS–PAGE, followed by immunoblotting with anti–S-tag and BiP antibodies. Inputs were also analyzed by immunoblotting with the same antibodies. AP, affinity purification. (B) Cells expressing NHK-HA were transfected with the indicated siRNA and the resulting WCE analyzed by SDS–PAGE and immunoblotting with the indicated antibodies. (C) Top, cells expressing NHK-HA and transfected with scrambled or Grp170 siRNA #1 or #2 in B were treated with cycloheximide for the indicated time and harvested, and the resulting WCE was analyzed with an anti HA antibody. Bottom, as for the top, except that cells were transfected with scrambled or Sil1 siRNA #1 or #2. (D) Parental Flp-In T-REx 293 cells were transfected with either scrambled or Grp170 siRNA #1. Where indicated, cells transfected with Grp170 siRNA #1 also stably expressed either siRNA-resistant Grp170-FLAG or G41L Grp170-FLAG. After siRNA transfection, cells were transfected with NHK-HA, treated with cyclohexamide for the indicated time, and harvested, and the resulting WCE was analyzed as in C. Bottom, as for the top, except that cells stably expressing FLAG-Sil1 were used.