Fig. 2
Identification of target proteins regulated by SUMO polymers. a Experimental set up for the identification of SENP6-regulated proteins. U2OS cells stably expressing His10-SUMO2 were infected with lentiviruses encoding shRNAs targeting SENP6 or a nontargeting control (ctrl) shRNA. Cells were lysed 3 days post infection and SUMOylated proteins were enriched by means of Ni-NTA pulldown. Enriched SUMOylated proteins were trypsin digested and prepared for label-free quantitative mass spectrometry. Peptides were identified by LC–MS/MS. The four experimental conditions of three biological replicates were analysed in two technical repeats per sample, resulting in a total of 24 MS runs. Black circles represent endogenous SUMO, yellow stars represent exogenous His10-SUMO2. b Immunoblot analysis of the three biological replicates analyzed by mass spectrometry. An antibody against SUMO2/3 was used to confirm efficient enrichment of SUMO conjugates and an increase of SUMO conjugates upon SENP6 knockdown. An antibody against SENP6 was used to confirm efficient knockdown. c Volcano plot showing all identified proteins within the SENP6 knockdown samples compared with the nontargeted control shRNA. Dashed lines indicate a cutoff at twofold change (log2 = 1) and a p-value of 0.05 (−log10 = 1.3), n = 3 independent experiments. SUMOylated proteins represented as blue circles were more abundant after SENP6 knockdown. The left panel shows identified centromere proteins and protein involved in centromere regulation represented by red circles, whereas the right panel shows DNA damage response proteins represented in red circles. Source data are provided as a Source Data file
