Figure 4.

PIAS1 and PIAS3 are required for sumoylation of MBD1 in vivo. (A) siRNA depletion of PIAS1, PIAS3 and both PIAS proteins in HeLa. GL2 is a control siRNA against firefly luciferase. (B) MBD1 and MBD1-SUMO1 detected in the extracts of PIAS-depleted cells (as in A). MBD1-SUMO1 is lost completely only when both PIAS1 and PIAS3 were depleted. (C) PCNA was used as a loading control for the experiments shown in (A) and (B). (D) HeLa cells were transfected with plasmids expressing the indicated proteins. PIAS1, GFP-PIAS1 and GFP-PIAS3 (both wild-type and catalytic mutants PIAS1^C350S and PIAS3^CS/HA) were detected with anti-PIAS1 and anti-GFP antibodies. (E) A Western blot of the same extracts as in (D) probed with anti-MBD1 antibodies demonstrates that MBD1-SUMO1 accumulates in cells transfected with GFP-PIAS1 and GFP-PIAS3 but not in cells transfected with catalytically inactive PIAS proteins. (F) MBD1 was immunoprecipitated from the same extracts as in (D) and (E), and MBD1-SUMO1 was detected with anti-Flag antibody.