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. 2006 Nov 13;27(2):622–632. doi: 10.1128/MCB.01160-06

FIG. 3.

FIG. 3.

PIAS1 stimulated myocardin sumoylation on a nonconsensus sumoylation site(s) via its RING domain. (A) Enhancement of myocardin sumoylation by PIAS1 via its RING domain was observed by Western blotting with lysates of HeLa cells transfected with indicated combinations of specified factors (PIAS1 or its RING mutant [mut]; 50 ng). Data are representative of two independent assays. (B) Ni-NTA pulldown followed by a protein blotting assay confirmed that PIAS1 potentiated myocardin sumoylation via transfected SUMO-1. (Upper panel) The blot was revealed by SUMO-1 antibody; (lower panel) the blot was revealed by V5 antibody. The asterisks inside the panels refer to the enhanced SUMO-1-V5-myocardin by PIAS1. (C) The K445R mutant was sumoylated in the presence of PIAS1. The asterisk inside the panel indicates the SUMO-1-conjugated K445R mutant. The results shown in panels B and C are representative of three independent assays. (D) Ni-NTA pulldown followed by a Western blotting assay confirmed that PIAS1 potentiated endogenous SUMO conjugation to myocardin. Analogous results were obtained from two independent assays. The asterisk inside the panel denotes the SUMO-conjugated myocardin. wt, wild type.