Using dot blot with immunochemical detection to evaluate global changes in SUMO-2/3 conjugation
Next, we examined the dependence of the Western blot and dot blot signal intensity on the percentage of conjugated SUMO-2/3 (Figure 2). To obtain defined levels of conjugated SUMO-2/3 in total cell lysates, the lysate from heat-shocked cells was mixed at various ratios with cell lysate of the same origin but treated with Ulp1 for global SUMO-2/3 deconjugation. The signal intensity of free SUMO-2/3 and SUMO-2/3 conjugates in the individual samples with comparable total protein content was verified by Western blot analysis (Figure 2A). The decrease of free SUMO-2/3 signal correlated with the theoretical increase of the level of SUMO-2/3 conjugates. However, for the increase of SUMO-2/3 conjugate, the signal was visible in the interval of 0% (only unconjugated SUMO-2/3) to approximately 60% (SUMO-2/3 conjugated on nitrocellulose), and up to 90% on PVDF (Figure 2). Dot blot analysis showed an approximately linear correleation between the signal and the level of conjugated SUMO-2/3 in the interval from 0% to 60% of SUMO-2/3 conjugated on nitrocellulose and 0 to 90% conjugated on PVDF, which was in agreement with results obtained by Western blot (Figure 2). Even with reduced protein quantities near the detection limit of dot blot, we observed a constant signal above 60%–70% of SUMO-2/3 conjugated on nitrocellulose membrane. Based on these results, we recommend using PVDF membranes for evaluation of global changes in SUMO-2/3 conjugation by dot blot.
Finally, we used dot blot to screen for changes in global SUMO-2/3 conjugation in HEK 293T cells grown under different conditions (Figure 3). Each sample was tested in four replicates using dot blot and the signal intensities were evaluated using Total Lab TL100 software and statistically processed (Figure 3). The results obtained from these dot blot analyses on PVDF membranes reflect the fact that treatment by MG132-induced SUMO-2/3 conjugate accumulation in a time dependent manner (Figure 3, MG132–1h and 2h), while treatment with the translational inhibitor cycloheximide strongly decreased the MG132-induced SUMO-2/3 conjugation. This negative effect of cycloheximide on MG132-induced conjugation of SUMO-2/3 in our assay agrees with previous observations (10, 17), where SUMO-2/3 deconjugation assays showed that cycloheximide treatment did not influence global cellular SUMO-2/3 levels, but instead affected SUMO-2/3 conjugation by blocking production of novel SUMOylation targets (17).
As shown above, our approach to evaluate global changes in cellular SUMO-2/3 conjugation is based on increased signal intensity of SUMO-2/3 upon its conjugation. We used this phenomenon to screen for changes in cellular SUMO-2/3 conjugation under different conditions by dot blot analysis on nitrocellulose and PVDF membrane, and showed that the PVDF membrane was more suitable for this application. When compared with Western blot, this technique offers the advantages of shorter time requirements, potential to screen large numbers of samples, and ease in evaluating the results using quantification software and statistical analysis. It should be noted that dot blot analysis can be used only to screen for changes in global cellular SUMO-2/3 conjugation; this method is not intended for evaluation of changes in SUMOylation of individual proteins. In those cases, Western blot analysis is still required. Dot blot is particularly useful for comparing SUMO conjugates contained in tissues (in individuals or under different pathological conditions), and screening for activators or inhibitors of SUMO-2/3 conjugation in cell cultures for potential development of drugs modulating SUMO-2/3 conjugation.
This work was funded by the Czech Ministry of Education Research Project grant MSM 6046137305 and by the Financial Support from Specific University Research (MSMT No. 21/2012).
The authors declare no competing interests.
Address correspondence to Zdeněk Knejzlík, Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 5, 16628 Prague, Czech Republic. Email: [email protected]">[email protected]
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