Figure 6.
PIAS1 and PIASxα do not enhance sumoylation of Dnmt3a in vivo or in vitro. (A) Two micrograms of full-length GAL4-Dnmt3a was transfected without (lane 1) or with 1.5 µg of SUMO-1 expression vector (lane 2) into COS-7 cells and whole cell extracts were prepared for western analysis with anti-GAL4-DBD antibody. In lanes 3 and 4, four micrograms of PIAS1 or PIASxα expression vector, respectively, are co-transfected with the SUMO-1 and GAL4-Dnmt3a expression vectors. (B) In vitro sumoylation reactions containing 35S-labeled-IVT Dnmt3a, performed as described in Figure 5B, were supplemented with recombinant GST-PIASxα (1, 5, 10 µg) and the reaction products analyzed on a 6% SDS–PAGE gel.