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

Figure 3.

Figure 3

PIASy enhances SUMO-conjugation in vivo. (A) Wild-type PIASy enhances, whereas mutant PIASy proteins abrogate, SUMO-conjugation. Wild-type, RING mutant, or Ser mutant T7-PIASy proteins were coexpressed with Flag-SUMO1 or Flag-SUMO2 in 293T cells. The RING mutant contains serine substitutions for cysteines 330, 335, and 340 and an alanine substitution for histidine 337 in the RING domain. The Ser mutant contains alanine substitutions for serines 470 to 474 in the Ser/Ac domain. Flag-SUMO–modified proteins from total sample buffer cell lysates were detected by an anti-Flag immunoblot (top). Numbers to the right indicate the molecular mass of protein markers in kilodaltons. The expression of T7-PIASy proteins in total cell lysates was determined by an anti-T7 immunoblot (bottom). (B) Characterization of mutant PIASy and LEF1 proteins. Wild-type or mutant LEF1 and T7-PIASy proteins were transiently expressed in 293T cells. Equivalent amounts of total cellular protein were immunoprecipitated with anti-T7 mAb. Coimmunoprecipitated LEF1 proteins were detected by an anti-LEF1 immunoblot (top). The LEF1 M2/K267R protein contains alanine substitutions for lysine 25, aspartate 26, and glutamate 27 (Hsu et al. 1998) and an arginine substitution for lysine 267 within the two consensus SUMO motifs. The expression of LEF1 or T7-PIASy proteins in total cell lysates was determined by anti-LEF1 (middle) or anti-T7 (bottom) immunoblots, respectively.