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. 2023 Aug 7;12:e88875. doi: 10.7554/eLife.88875

Figure 4. Hsp31 is a potent scavenger of methylglyoxal.

Figure 4.

(A) In vitro methylglyoxalase activity. Purified Hsp31 members 5 µg each were incubated with 0.5 mM MG, and the absorbance at 530 nm was monitored. (B) Spot assay. Respective strains grown till the mid-log phase were harvested and treated with 10 mM MG for 5 hr before spotting on SD Leu- plates. Images were captured at 36 hr. (C–E) Hsp31 reduces MG-derived AGE modifications. Individual strains were treated with 10 mM MG in the culture tubes for 12 hr, and the macromolecule glycation was analyzed using an anti-MAGE antibody. (F) C138 amino acid residue is critical for methylglyoxalase activity. 5 µg of Hsp31-WT and Hsp31C138A mutant were incubated with MG, and the activity was examined at 530 nm. BSA was used as negative control in enzymatic assay. Glycation levels represent the relative intensities of the dots and lanes compared with WT. One-way ANOVA with Dunnett’s multiple comparisons test was used to determine significance from three independent biological replicates, *, p≤0.05; **, p≤0.01; ***, p≤0.001; NS, not significant.

Figure 4—source data 1. Source data for Figure 4A contains densitometric values for graph.
Figure 4—source data 2. Source data for Figure 4C contains raw image of western blot and densitometric values for graph.
Figure 4—source data 3. Source data for Figure 4D contains raw image of western blot and densitometric values for graph.
Figure 4—source data 4. Source data for Figure 4E contains raw image of western blot and densitometric values for graph.
Figure 4—source data 5. Source data for Figure 4F densitometric values for graph.