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. 2001 Dec 1;15(23):3088–3103. doi: 10.1101/gad.944801

Figure 7.

Figure 7

(A) PIASy relocalizes LEF1 to nuclear bodies. COS7 cells were either mock transfected or were transfected with expression constructs encoding for LEF1 or T7-PIASy. The intracellular distribution of the indicated proteins was detected by indirect immunofluorescence and analyzed by confocal microscopy. The intracellular distribution of LEF1 and T7-PIASy was detected with anti-LEF1 antibody and anti-T7 mAb, respectively. LEF1 alone displays a homogeneous distribution throughout the nucleus (upper, middle panel) but is detected in punctate nuclear bodies in the presence of T7-PIASy (middle, lower panels). Approximately 20% of the cells showed colocalization of LEF1 and T7-PIASy in <20 nuclear bodies (middle panels),whereas >50% of the cells showed colocalization of LEF1 and T7-PIASy in multiple (>20) nuclear bodies (lower panels). (B) PIASy colocalizes with SUMO-modified proteins. COS7 cells were transfected with expression constructs encoding for wt T7-PIASy, T7-PIASy RING mut, Flag-SUMO1, Flag-SUMO2, wt LEF1, or mut LEF1 M2/K267R, as indicated. PIASy in the upper panels was detected with anti-PIASy antibody. The T7-PIASy proteins in the remaining panels were detected with anti-T7 mAb. Flag-SUMO1 was detected with anti-Flag mAb. T7-PIASy efficiently colocalized with Flag-SUMO1 or Flag-SUMO2 in nuclear bodies in >80% of cotransfected cells (top panels; data not shown). Coexpression of Flag-SUMO1 or Flag-SUMO2 with LEF1 and T7-PIASy enhanced nuclear body localization of LEF1 by approximately twofold (second panels; data not shown). The T7-PIASy RING mutant protein displays homogeneous staining throughout the nucleus and is unable to relocalize LEF1 to nuclear bodies (third panels). The LEF1 M2/K267R mutant protein is efficiently relocalized to nuclear bodies in the presence of T7-PIASy and Flag-SUMO2 (bottom panels). (C) PIASy partially colocalizes with PML bodies. T7-PIASy, LEF1, and epitope-tagged HA-Sp100 were transfected into COS7 cells, as indicated. The intracellular distributions of T7-PIASy and dimethyl-lysine 9 of histone H3, a marker for heterochromatin, were detected with anti-T7 mAb and anti-MeK9, respectively (left). The distribution of LEF1 (coexpressed with T7-PIASy) and SC35, a component of the splicing machinery, was detected with anti-LEF1 and anti-SC35 mAb, respectively (center). The distribution of LEF1 (coexpressed with T7-PIASy) and HA-Sp100, a component of PML nuclear bodies, was detected with anti-LEF1 antibody and anti-HA mAb, respectively (right). LEF1 only showed a partially overlapping pattern of distribution with HA-Sp100 in PML nuclear bodies.