Fig. 2.
Identification of heat shock-inducible SUMO-2 substrates. A, SUMO-2 substrates were immunoprecipitated from untreated and heat-shocked K562HA-SUMO-2 and heat-shocked parental K562 cells. The immunoprecipitated proteins were desumoylated on beads with recombinant Ulp-1, separated by SDS-PAGE, silver-stained, and identified by LC-MS/MS. B, an aliquot from the immunoprecipitated and desumoylated samples was analyzed by Western blotting with antibodies for topoisomerase I (TopoI), DDX21, and PARP-1. C, PARP-1 undergoes SUMO-2 modification in a heat shock-inducible manner. HeLa cells transfected with Myc-tagged PARP-1 and SUMO-2 were exposed to heat shock at 43 °C for 2 h. Cells were lysed under denaturing conditions, renatured, sonicated, and centrifuged. α-Myc immunoprecipitates (IP) were separated by SDS-PAGE and blotted against SUMO-2/3.