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. 2015 Jan 15;26(2):185–194. doi: 10.1091/mbc.E14-07-1202

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© 2015 Mehnert, Sommermeyer, et al. 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 5: — Accumulation of substrate at Hrd3 in cells lacking Scj1. (A) HA-Hrd3 expressed from the chromosomal locus was purified from lysates of yeast cells of the given genotype and the precipitates analyzed by immunoblotting. (B) HA-Hrd3 or HA-Hrd3KR expressed from plasmids in cells deleted for hrd3 was purified from lysates prepared under nondenaturing conditions and the precipitates analyzed by immunoblotting with the given antibodies. (C) Pulse- chase experiment to determine the degradation of CPY* in yeast cells of the indicated genotype. Mean values of three independent experiments with SD of the mean. (D) Lysates were prepared from cells lacking Hrd3 and containing plasmids for the expression of Hrd3 or Hrd3KR. The HA-tagged Hrd3 variants were then immunoprecipitated and the precipitates analyzed by immunoblotting. Where indicated, CPY* was overexpressed using a multi-copy-number plasmid.