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. 2017 Apr 3;16(6):1081–1097. doi: 10.1074/mcp.M116.066159

Fig. 5.

Fig. 5.

Validation of potential identified SUMOylated targets with recombinant SUMOylation system in E. coli and in eukaryotic cells. The mass spectrometry SUMO-identified proteins: A, TRIM63, TRIM54. B, myosin-ATPase domain (Myo-ATPase). C, aspartate aminotransferase (AATM), ornithine aminotransferase (OAT), ATP synthase subunit-ε (ATP5E),and ATP synthase subunit-α (ATP5A). D, calsequestrin 1 (CASQ1), calsequestrin 2 (CASQ2), and Triadin (cTRDN) luminal C-terminal portion, tagged with GST, were expressed in BL21 bacteria together with pSUMO1 (lane 1), pSUMO2 (lane 2), pSUMO3 (lane 3) vectors containing the SUMOylation system described under “Experimental Procedures,” and empty vector (mock, M). Bacteria lysates were probed with anti-GST antibodies to detect the expression of not modified protein indicated with an arrow (lane M), and the corresponding SUMOylated bands appeared as low speed bands above the native GST-tagged protein. Specific antibodies raised against native proteins (TRIM63 and ATP5A) were used to validate the results obtained with anti-GST antibodies. E, candidates were co-expressed in eukaryotic cells with different amounts of the recombinant SUMO E2 ligase, Ubc9. SUMOylated bands appeared as new low migrating bands in presence of Ubc9. Square brackets indicate poly-SUMOylation of the recombinant proteins expressed both in bacteria and in eukaryotic cells. All blots are representative of three independent experiments performed in triplicate.