Chimeric E3-dependent sumoylation of human Smad4.

<p>Western blot analysis of cell lysate prepared from DH5α-Z1 cells expressing human Smad4 in the presence (+) or absence (-) of different SUMO-conjugation cascade components. The E1 and E2 enzymes were expressed from either the medium copy plasmid pZA31-SMCS (++) or the low-copy plasmid pZS31-SMCS (+). PIASxβ (lane 4) as well as a panel of E3 variants (lanes 5–8; see Fig. 1c caption for details) were tested for functionality. Smad4 was detected using anti-FLAG antibodies (a), while SUMO-1 was detected using anti-SUMO-1 antibodies (b). Detection of endogenous DnaK with anti-DnaK antibodies served as a loading control.