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. 2016 Sep 1;27(17):2735–2741. doi: 10.1091/mbc.E16-03-0192

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© 2016 Howard and Tansey. 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 4: — Mutation of Cdc48 suppresses the effect of proteasome inhibition on Gcn4 activity. (A) GCN4-HA (GHY356) yeast carrying a copper-inducible His-Ub expression plasmid (pUB221) were grown to log phase in minimal medium and treated with CuSO₄ and DMSO or MG132 for 1 h. Yeast were induced with SM or DMSO for an additional 1.5 h, at which time protein lysates were collected under denaturing conditions. Ubiquitin-conjugates were captured by nickel-resin (Ni-NTA) chromatography, resolved by SDS–PAGE, and probed for HA-tagged Gcn4 protein by Western blotting. A sample of the input material to the nickel resin was also probed for HA-tagged Gcn4. IB, immunoblot. (B) GCN4 CDC48-MYC (GHY285), GCN4-HA CDC48 (GHY025), GCN4-HA CDC48-MYC (GHY287), K0-GCN4-HA CDC48 (GHY124), and K0-GCN4-HA CDC48-MYC (GHY293) yeast were grown to log phase at 30°C in minimal medium and treated with DMSO or MG132. After 1 h, Gcn4 was induced with SM for 1.5 h. Protein lysates were collected and Gcn4-HA immunoprecipitated (IP) via an anti-HA antibody, and IPs were probed with antibodies against the Myc-epitope (Cdc48) and HA-epitope (Gcn4) tags. A sample of the input material to the IP was also probed for Myc-tagged Cdc48. (C, D). CDC48 (RHY2455) and cdc48-3 (RHY2457) strains were grown to log phase at 30°C in minimal medium and treated with DMSO or MG132. After 1 h, strains were treated with DMSO or SM for 1.5 h, RNA harvested, and processed as described to measure mRNA levels from the ARG1 (C) and HIS4 (D) loci. n = 4. (E, F). CDC48 GCN4-HA (GHY116) and cdc48-3 GCN4-HA (GHY118) strains were grown to log phase at 30°C in minimal medium, treated with either DMSO or MG132 for 1 h, and induced with SM for 1.5 h. ChIP was then performed using antibodies against the HA-epitope tag. Coprecipitating ARG1 (E) or HIS4 (F) promoter DNAs were quantified by qPCR, expressed relative to the percentage of input DNA. n = 3. (G) CDC48 (RHY2455), cdc48-3 (RHY2457), and cdc48-3::CDC48 (GHY279) strains were grown and treated as described in C and RNA harvested, and RT-qPCR was performed to quantify ARG1 transcripts. n = 3. (H) CDC48 GAL80 (RHY2455), cdc48-3 GAL80 (RHY2457), CDC48 gal80 (GHY304), and cdc48-3 gal80 (GHY305) yeast were grown in raffinose medium and treated with water or 2% galactose for 1.5 h. RNA was collected and used for RT-qPCR for GAL10. Each qPCR was internally normalized to ACT1 and then normalized to the galactose-treated CDC48 GAL80 sample. Error bars represent SEM. n = 3.