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. 2020 Nov 27;25(23):5571. doi: 10.3390/molecules25235571

Figure 2.

Figure 2

Experimental Setup and workflow of SUMO immunoprecipitation and mass spectrometry. (A) Experimental Setup. Mice underwent cardiac surgery in order to ligate left anterior descending artery for 30 min (Isch) and re-open it again for 1 h (I/R1) or 24 h (I/R24) reperfusion. Four mouse hearts for each condition (Sham, Isch, I/R1 or I/R24) were pooled as one biological replicate. The same replicate was used for both SUMO and IgG Control IP. All experiments were performed in triplicates. (B) Experimental Workflow of SUMO immunoprecipitation and mass spectrometry. Mouse hearts were snap frozen and pulverized in liquid nitrogen. Powder was lysed in 1% SDS buffer and homogenized in a ball mill. IPs were performed using in-house produced SUMO1-, SUMO2/3- or IgG antibody coupled beads. An aliquot of IP input was used for proteome analysis. IP Eluates representing SUMO1 or SUMO2/3 proteome were separated by SDS-PAGE and subjected to in-gel digestion protocol using trypsin. Peptides were analyzed by a streamlined in-house LC-MS/MS Bioinformatics pipeline.