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. 2005 May;25(9):3506–3518. doi: 10.1128/MCB.25.9.3506-3518.2005

FIG. 2.

FIG. 2.

Sumoylation of Tcf-4 by PIASyK35R. (A) Ability of PIASyK35R to enhance sumoylation. 293 cells expressing the indicated proteins were incubated with 10% TCA, and the precipitates were probed with the anti-HA and anti-Flag antibodies. IB, immunoblotting; Ab, antibody; WT, wild type; CA, PIASy RING mutant; ΔGG, SUMO-1-(1-95). (B) Sumoylation of Tcf-4 in vitro. GST-Tcf-4 was incubated with GST-Aos1/His6-Uba2, GST-SUMO-1(GG), His6-Ubc9, and MBP-PIASy, or MBP-PIASyK35R. After the incubation, the mixtures were probed with the anti-Tcf-4 antibody. Arrowheads indicate GST-Tcf-4 modified with GST-SUMO-1, and the arrow indicates GST-Tcf-4. (C) Sumoylation of Tcf-4 in intact cells. Precipitates were prepared from 293 cells expressing the indicated proteins by adding TCA and probed with the anti-Tcf-4 and anti-Flag antibodies. The arrow indicates Tcf-4. The results shown are representative of three independent experiments. (D) Interaction of Tcf-4 with PIASy. The lysates (20 μg of protein) of COS cells expressing the indicated proteins were probed with the anti-HA and anti-Flag antibodies (lanes 1 to 4). The same lysates (200 μg of protein) were immunoprecipitated with the anti-Flag antibody, and the immunoprecipitates were probed with the anti-HA and anti-Flag antibodies (lanes 5 to 7).