Autoradiograms showing 32P-UBL thioesters after E2 charging by E1~32P-UBLs (top) and reduction with 100 mM DTT (bottom): A, NAE1-UBA3~NEDD8; B, UBA1~Ub; C, SAE1-UBA2~SUMO1; D, UBA7~ISG15; E, UBA6~Ub; F, UBA6~ FAT10. G, Immunoblot showing 100 mM DTT reduction of E2~NEDD8 complex detected with antibodies against E2 (α-Flag) and NEDD8 (α-N8) from NIH 3T3 cells infected with indicated retroviruses. H, Immunoblot as in G (top) from whole cell extracts (WCE) of NIH 3T3 cells infected with indicated retroviruses and lysed in urea buffer, and from nickel-agarose pull-downs (His). * expected MW of E2~NEDD8 oxy-ester complex.