Fig. 6

SUMOylated NPM1 directly binds to the RAP80 SUMO-interacting motif (SIM) domain. a HEK293T cells transfected with Flag-NPM1 WT or K263R mutant vectors were treated with or without IR, and then subjected to co-IP analysis using anti-RAP80 and anti-Flag antibodies. b U2OS cells transfected with Flag-NPM1 WT or K263R were grown on collagen-coated microchamber slides. After fixation, in situ PLA for NPM1 WT or K263R and RAP80 was performed with anti-Flag and anti-RAP80 antibodies. The PLA-detected proximity (PROX) complexes are represented by the fluorescent rolling circle products (red dots). Scale bar, 10 μm. c Quantification of the PROX dots per cell is shown as mean ± SEM with the P value indicated. ***P < 0.001. d HEK293T cells transfected with Flag-RAP80 FL or ΔSIM mutant vectors were treated with or without IR, and then subjected to co-IP analysis using anti-NPM1 and anti-Flag antibodies. e In vitro interaction of purified RAP80 with NPM1-SUMO2/3. The full-length and SIM domain (40–47 aa) deleted truncation of His-RAP80 and GST-NPM1 were purified from E. coli. GST-NPM1 protein was SUMOylated in vitro, and the pull-down analysis was performed. f Schematic of the direct interaction between SUMOylated NPM1 and RAP80 SUMO-interacting motif (SIM). Unprocessed scans of blots are provided in Supplementary Fig. 13