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. 2018 Apr 15;29(8):948–963. doi: 10.1091/mbc.E17-11-0652

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© 2018 Kama et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 5: — Cdc48 and the ubiquilins reduce the detergent-insoluble fraction of Cps1. (A) The detergent-insoluble fraction of Cps1 decreases substantially in WT but less so cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ cells expressing endogenous myc-Cps1 from its genomic locus were subjected to a cycloheximide (CHX)-chase degradation/sedimentation assay to resolve the amount of Triton X-100 detergent-insoluble Cps1 in the pellet fraction, as described under Materials and Methods. Samples of were removed after 0, 15, and 30 min of CHX treatment before processing to determine the relative amounts of myc-Cps1 in the total, pellet, and supernatant (see Supplemental Figure S6A) fractions by Western analysis with anti-myc antibodies. Anti-Snf7 antibodies were employed to assess the levels of total protein loaded and in subsequent quantitative analyses used for normalization of the results (unlike actin, Snf7 is not degraded upon CHX treatment). The amount of insoluble Cps1 in the pellet was normalized to the loading control, after which the percentage was calculated relative to the amount at 0 h. A representative experiment is shown in the top panel. A histogram of the quantification of three repetitive experiments is shown beneath. kDa = kilodaltons. (B) The detergent-insoluble fraction of eisosome protein, Pil1, does not change in cdc48-10 ddi1Δ cells. WT and cdc48-10 ddi1Δ yeast expressing myc-Pil1 from its genomic locus were subjected to the cycloheximide-chase degradation/sedimentation assay in A to resolve the amount of Triton X-100 detergent-insoluble Pil1 by Western analysis using anti-myc antibodies as in A. A histogram of showing the results of three repetitive experiments is shown beneath. (C) The level of NP-40 detergent-insoluble Cps1 increases in ddi1Δ and ddi1Δ dsk2Δ rad23Δ cells. WT control cells (W303), ddi1Δ, and ddi1Δ dsk2Δ rad23Δ cells expressing HA-Cps1 were subjected to the same procedure as in A, and the percentage of insoluble Cps1 in the pellet fraction after 1 h was calculated after the normalization for gel loading. In the representative experiment, the level of NP-40-insoluble Cps1 increased in the ddi1Δ and ddi1Δ dsk2Δ rad23Δ pellet fractions by 34 and 78%, respectively. A histogram of three repetitive experiments is shown at the bottom.