(A) U2OS or 6xHis-SUMO2 U2OS cells were synchronized in different stages of the cell cycle. SUMO2-modified proteins were purified by Ni-NTA agarose chromatography and analyzed by SDS-PAGE and immunoblotting with antibodies against APC4 or SUMO2/3. Single asterisks (*) indicates SUMO modified forms of APC4. A double asterisk (**) indicates unconjugated 6xHis-SUMO2. (B) Wild type and mutant APC4 proteins were expressed in rabbit reticulocyte lysate in the presence of [35S]-methionine and incubated for the indicated times in modification reactions containing SUMO E1 and E2 enzymes and SUMO1. Reactions were analyzed by SDS-PAGE and autoradiography. Asterisks (*) indicate sumoylated forms of APC4. (C) Endogenous APC4 was depleted by siRNA, and APC4 WT or APC4KR were induced by doxycycline for 48 hr. Immunoprecipitations were performed with an antibody against APC4, followed by immunoblotting for APC4 or SUMO1. Asterisks indicate sumoylated APC4. (D) HeLa cells were transfected with control, SENP1 or SENP2 specific siRNAs. 36 hr following transfection, cells were treated with 2 mM thymidine for 19 hr, released into nocodazole or 6 hr, followed by treatment with MG132 for 2 hr to synchronize cells in metaphase. Whole cell lysates were immunoblotted with antibodies specific for APC4, SENP1, SENP2, and GAPDH. Arrows indicate sumoylated APC4.