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. 2004 Jun 1;101(24):8870–8875. doi: 10.1073/pnas.0308605101

Fig. 2.

Fig. 2.

GATA-1 is sumoylated in vitro and in vivo.(A) In vitro sumoylation of GATA-1. Radioactively labeled in vitro translated GATA-1 was incubated in the presence of recombinant components of the SUMO conjugation pathway as indicated. Two isoforms of GATA-1 are visible (see text). GST-SUMOΔ6 is a C-terminal truncation lacking the glycine residues required for attachment to substrates. Reactions were separated by SDS/PAGE and visualized by autoradiography. (B) Sumoylation of GATA-1 in 293T cells. WT GATA-1 or the K137R mutant were transfected in 293T cells with or without a vector expressing GFP-SUMO-1. Lysates were separated by SDS/PAGE, and GATA-1 was detected by immunoblotting. (C) Sumoylation of GATA-1 in osteosarcoma cells. WT GATA-1 or the K137R mutant were transfected in MG63 cells together with GFP-SUMO-1. Lysates were immunoprecipitated with a monoclonal antibody to GATA-1 and immunoblotted with an antibody to SUMO-1 (Left). IgG heavy chains are indicated. GATA-1 and GFP-SUMO-1 were also detected in total lysates (Right). The asterisk indicates a nonspecific band recognized by the GATA-1 antibody in these cells. (D) Sumoylation of GATA-1 in mouse erythroleukemia cells. Lysates from murine erythroleukemia cells were immunoprecipitated with an antibody to SUMO-1 and immunoblotted with an antibody to GATA-1. Immunoprecipitation with an unrelated monoclonal antibody served as a negative control.