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Using dot blot with immunochemical detection to evaluate global changes in SUMO-2/3 conjugation
Markéta Častorálová, Tomáš Ruml, and Zdeněk Knejzlík
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Figure 1.

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).

Competing interests

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]

1.) Tatham, M.H., S. Kim, B. Yu, E. Jaffray, J. Song, J. Zheng, M.S. Rodriguez, R.T. Hay, and Y. Chen. 2003. Role of an N-terminal site of Ubc9 in SUMO-1,-2, and-3 binding and conjugation. Biochemistry 42:9959-9969.

2.) Johnson, E.S. 2004. Protein modification by SUMO. Annu. Rev. Biochem. 73:355-382.

3.) Kim, J.H., and S.H. Baek. 2009. Emerging roles of desumoylating enzymes. Biochim. Biophys. Acta 1792:155-162.

4.) Geiss-Friedlander, R., and F. Melchior. 2007. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell Biol. 8:947-956.

5.) Saitoh, H., and J. Hinchey. 2000. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J. Biol. Chem. 275:6252-6258.

6.) Zhou, W., J.J. Ryan, and H. Zhou. 2004. Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses. J. Biol. Chem. 279:32262-32268.

7.) Uzunova, K., K. Gottsche, M. Miteva, S.R. Weisshaar, C. Glanemann, M. Schnellhardt, M. Niessen, H. Scheel. 2007. Ubiquitin-dependent proteolytic control of SUMO conjugates. J. Biol. Chem. 282:34167-34175.

8.) Golebiowski, F., I. Matic, M.H. Tatham, C. Cole, Y. Yin, A. Nakamura, J. Cox, G.J. Barton. 2009. System-wide changes to SUMO modifications in response to heat shock. Sci. Signal. 2:ra24.

9.) Schimmel, J., K.M. Larsen, I. Matic, M. van Hagen, J. Cox, M. Mann, J.S. Andersen, and A.C. Vertegaal. 2008. The ubiquitin-proteasome system is a key component of the SUMO-2/3 cycle. Mol. Cell. Proteomics 7:2107-2122.

10.) Tatham, M.H., I. Matic, M. Mann, and R.T. Hay. 2011. Comparative proteomic analysis identifies a role for SUMO in protein quality control. Sci. Signal. 4:rs4.

11.) Lee, Y.J., and J.M. Hallenbeck. 2006. Insights into cytoprotection from ground squirrel hibernation, a natural model of tolerance to profound brain oligaemia. Biochem. Soc. Trans. 34:1295-1298.

12.) Lee, Y.J., S. Miyake, H. Wakita, D.C. McMullen, Y. Azuma, S. Auh, and J.M. Hallenbeck. 2007. Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells. J. Cereb. Blood Flow Metab. 27:950-962.

13.) Yang, W., Q. Ma, G.B. Mackensen, and W. Paschen. 2009. Deep hypothermia markedly activates the small ubiquitin-like modifier conjugation pathway; implications for the fate of cells exposed to transient deep hypothermic cardiopulmonary bypass. J. Cereb. Blood Flow Metab. 29:886-890.

14.) Yang, W., H. Sheng, D.S. Warner, and W. Paschen. 2008. Transient global cerebral ischemia induces a massive increase in protein sumoylation. J. Cereb. Blood Flow Metab. 28:269-279.

15.) Datwyler, A.L., G. Lättig-Tünnemann, W. Yang, W. Paschen, S.L. Lee, U. Dirnagl, M. Endres, and C. Harms. 2011. SUMO2/3 conjugation is an endogenous neuroprotective mechanism. J. Cereb. Blood Flow Metab. 31:2152-2159.

16.) Cimarosti, H., E. Ashikaga, N. Jaafari, L. Dearden, P. Rubin, K.A. Wilkinson, and J.M. Henley. 2012. Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones. J. Cereb. Blood Flow Metab. 32:17-22.

17.) Častorálová, M., D. Březinová, M. Švéda, J. Lipov, T. Ruml, and Z. Knejzlík. 2012. SUMO-2/3 conjugates accumulating under heat shock or MG132 treatment result largely from new protein synthesis. Biochim. Biophys. Acta 1823:911-919.

18.) Vertegaal, A.C., S.C. Ogg, E. Jaffray, M.S. Rodriguez, R.T. Hay, J.S. Andersen, M. Mann, and A.I. Lamond. 2004. A proteomic study of SUMO-2 target proteins. J. Biol. Chem. 279:33791-33798.

19.) Vertegaal, A.C., J.S. Andersen, S.C. Ogg, R.T. Hay, M. Mann, and A.I. Lamond. 2006. Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol. Cell. Proteomics 5:2298-2310.

20.) Ribet, D., M. Hamon, E. Gouin, M.A. Nahori, F. Impen, H. Neyret-Kahn, K. Gevaert, J. Vandekerckhove. 2010. Listeria monocytogenes impairs SUMOylation for efficient infection. Nature 464:1192-1195.

21.) Jones, M.C., L. Fusi, J.H. Higham, H. Abdel-Hafiz, K.B. Horwitz, E.W. Lam, and J.J. Brosens. 2006. Regulation of the SUMO pathway sensitizes differentiating human endometrial stromal cells to progesterone. Proc. Natl. Acad. Sci. USA 103:16272-16277.

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