Fig. 1

Identification of SAP130 as an FAF1-interacting protein and a SUMO substrate. A Schematic representation of the SAP130 clone (F23) that interacted with LexA-FAF1 bait, including the positions of three potential SUMO conjugation sites. B Yeast co-transformed as indicated with bait and prey were analyzed with quantitative β-Gal assays and on X-Gal-containing plates. LexA-lamin served as a negative control. C COS-1 cells were transiently transfected with the indicated combinations of expression constructs encoding Flag-tagged FAF1 and HA-tagged SAP130, respectively. After 48 h, whole cell lysates (WCL) were used for immunoprecipitation (IP), followed by Western blot (WB) analysis with the indicated antibodies. The arrow indicates the position of the co-precipitated FAF1. Star indicates the heavy chain of IgG. D SAP130 is sumoylated in vivo. A plasmid expressing HA-tagged SAP130 was co-transfected with EGFP control or EGFP-SUMO1 plasmid into COS-1 cells. Cell lysates were used for IP with an anti-HA antibody, followed by WB analysis using antibodies against HA and GFP. The black asterisk and white asterisks indicate EGFP-SUMO-1-modified and unmodified SAP130 proteins, respectively. The blue and red arrowheads indicate the EGFP and EGFP-SUMO1 proteins, respectively. E SUMO1 modification of SAP130 occurs at Lys-794, 878, and 932. HA-tagged wild-type SAP130 and various mutant SAP130 proteins were subjected to an in vitro sumoylation assay as described in Materials and Methods. SUMO-1 modified and unmodified SAP130 proteins were analyzed by immunoblotting with anti-HA antibody. The bracket and arrow indicate SUMO-modified and unmodified SAP130 proteins, respectively. F In vivo sumoylation analyses of wild-type and mutants of SAP130. The assays were performed as described in Fig. 1D. The bracket and arrow indicate EGFP-SUMO-1-modified and unmodified SAP130 proteins, respectively. The blue and red arrowheads indicate the EGFP and EGFP-SUMO1 proteins, respectively. The immunoblots were cropped for clarity. Full length blots are presented in Supplemental Figure S8