And strains were labeled with S-Met for 5 min at 30°C, and rates of degradation were measured at 30 or 38°C as in

To measure the degradation of oxidant-damaged proteins, WT and strains were exposed to paraquat or cadmium for 90 min, followed by labeling with S-Met for 5 min, and rates of degradation were measured as in .<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

(A) WT, , and strains were labeled with S-Met for 5 min, washed, and shifted to 38°C or treated with paraquat at 30°C

At the indicated times, lysates were prepared, and equal amounts of lysate proteins were subjected to differential centrifugation. The amounts of labeled protein present in pellets obtained by centrifuging for 20 min at 9,300 and by ultracentrifugation at 275,000 for 60 min were measured. To determine the amounts of labeled long-lived proteins that accumulate in these fractions, cells were shifted to 38°C or exposed to paraquat 3 h after labeling. (B) , , and strains were labeled with S-Met for 5 min at 30°C. The amounts of radiolabeled protein present in 275,000 pellet were measured as described in . (C) Yeast were grown exponentially and treated with paraquat as in . Cell lysates were subjected to centrifugation and the presence of carbonylated proteins in the 275,000 pellet assayed by OxyBlot as in .<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

(A) and mutants were grown exponentially at 30°C and treated with paraquat or cadmium for 90 min

Then cell proteins were labeled with S-Met for 5 min, and rates of degradation measured as in . (B) and mutants were grown exponentially, and a portion was exposed to paraquat for 90 min. The presence of carbonylated proteins in equal amounts of cell extracts was assayed by OxyBlot as in . To visualize oxidant damage in the control cells, approximately three times more protein was loaded than in cells treated with paraquat.<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

(A) and strains were labeled with S-Met for 5 min at 30°C, and rates of degradation at 30 or 38°C were measured as in

To measure the degradation of oxidant-damaged proteins, and strains were exposed to paraquat or cadmium for 90 min, followed by labeling with S-Met for 5 min, and rates of degradation were measured as in . (B) and the mutant were grown exponentially, and a portion was exposed to paraquat for 90 min. The presence of carbonylated proteins in equal amounts of cell proteins was assayed by OxyBlot as in .<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

(A) Proteins were labeled with S-Met for 5 min at 30°C to label recently synthesized proteins or 90 min to label long-lived proteins

After washing twice with chase media containing cyclohexamide, the cells were shifted to the indicated temperatures and rates of degradation were measured. The data shown in this and the subsequent experiments represent the means ± SD obtained from three experiments. (B) Cell proteins were labeled with S-Met for 5 min, washed, resuspended in chase media, and shifted to 38°C immediately or 30, 60, or 90 min later. The amounts of protein degradation after 1 h were measured. Degradation rates after 2 or 3 h showed similar differences as after 1 h (not depicted).<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

Cells were labeled with S-Met for 5 min at 30°C and shifted to 38°C or maintained at 30°C

Rates of degradation were measured as in .<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

And strains were labeled with S-Met for 5 min at 30°C, and rates of degradation at 30 or 38°C and upon exposure to paraquat or cadmium were measured as in

<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

(A) Yeast were grown exponentially, and a portion of cells were treated with cycloheximide for 2 h to allow the degradation of short-lived proteins (the times studied in )

Both the control and treated cells were then exposed to paraquat for 90 or 150 min. The presence of carbonylated proteins in equal amounts of cell proteins was assayed after derivitization with DNP-hydrazine (DNPH) and then Western blotting with an anti–DNP-hydrazone antibody. A control lane without the treatments with DNPH and paraquat is included to show the specificity of the antibody. Equal loading of lanes was shown with an eIF5A antibody. (B) WT, mutant, and the mutant expressing from a plasmid were labeled with S-Met for 5 min at 30°C. Rates of protein degradation were measured at 30°C as in .<p><b>Copyright information:</b></p><p>Taken from "Heat shock and oxygen radicals stimulate ubiquitin-dependent degradation mainly of newly synthesized proteins"</p><p></p><p>The Journal of Cell Biology 2008;182(4):663-673.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518706.</p><p></p

N-terminal specificity of decapeptide trimming by ERAP1.

<p>All peptides are based on the same template varying in their N-terminus (indicated as X in the sequence below the graph). 100 µM of each peptide was incubated with 40 ng of ERAP1 at 37°C and the reaction products analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003658#pone-0003658-g001" target="_blank">figure 1</a>. A representative experiment is shown here. Peptides carrying hydrophobic residues at their N-termini were trimmed fastest, whereas peptides carrying charged (R, K or E) or hydrophilic (T) residues in their N-termini were more resistant to cleavage by ERAP1.</p

Trimming of the N-terminal leucine of N- and C- extended versions of LSIINFEKL peptide.

<p><i>Panels</i> A–C, typical C18 chromatograms comparing the trimming for LSIINFEKL and the C- and N-terminal extended versions LSIINFEKLAAAL and LAAALSIINFEKL. In each panel two chromatograms are shown, the top one in the absence of enzyme and the bottom in the presence of 40 ng ERAP1. All incubations were done for 1 hr at 37°C. Black arrows indicate new peaks detected after incubation with the enzyme. Under these conditions LSIINFEKL and LSIINFEKLAAAL are trimmed about to the same extent and LAAALSIINFEKL is resistant to any trimming. <i>Panel</i> D, ERAP1 trimming rates calculated as the average of 3 to 5 experiments. Because of the large difference between trimming rates the y-axis is logarithmic for better visualization.</p