FIG. 2.

Sumoylation of p45/NF-E2 in vivo. (A) Sumoylation of p45 in transfected K562 cells. The transfections of K562 cells were carried out using pEF-p45 and pCMV-His-SUMO-1 as described in Materials and Methods. The whole-cell extracts were then prepared and incubated with Ni-NTA agarose to isolate His-tagged protein(s). The retained fractions were analyzed by immunoblotting (IB) with anti-p45. (B) Requirement of K368 for sumoylation of human p45/NF-E2 in vivo. 293 cells were transfected with different combinations of the expression plasmids. The whole-cell extracts (WCE) were then prepared, IP with anti-Flag, and immunoblotted (IB) with anti-p45. Note that sumoylated p45 could only be detected in the 293 extract containing myc-p45 and FlagDsRed-SUMO-1 GG (lane 1, upper panel). The WCE prior to IP were also analyzed by Western blotting with anti-p45 (middle panel) and anti-SUMO-1 (lower panel), respectively. The arrowheads point to the positions of the sumoylated p45 on the gel. (C) Sumoylation of endogenous p45 in K562 cells. The nuclear extract was prepared from K562 cells, fractionated by precipitation with 20% to 50% ammonium sulfate, and analyzed by immunoblotting with anti-p45 (left panel) or with anti-SUMO-1 (middle panel). The 20% ammonium sulfate fraction was also immunoprecipitated with rabbit preimmune serum (left lane, right panel) or rabbit anti-p45 (right lane, right panel) and then hybridized with mouse anti-SUMO-1. (D) Sumoylation of endogenous p45 in mouse E14.5 fetal liver. The whole-cell extract was prepared from mouse E14.5 fetal livers as described in Materials and Methods and then analyzed by Western blotting using anti-p45 (lane 1) or anti-SUMO-1 (lane 2) as the probe. The extract was also immunoprecipitated with rabbit preimmune serum (lane 3) or rabbit anti-p45 (lane 4). The immunoprecipitates were then analyzed by Western blotting using mouse anti-SUMO-1 antibody as the probe.