Fig. 2.

Glis3 is SUMOylated by PIAS-family proteins. A. BRIN BD11 or HEK293T cells were transfected with FLAG empty vector or FLAG Glis3 along with HA-SUMO1, Myc Ubc9, Myc PIAS1, or Myc PIASy as indicated. After 48 h, co-immunoprecipitation was performed using a rat anti-HA antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-FLAG M2 or anti-Myc primary and goat anti-mouse-HRP secondary antibodies. B. HEK293T cells were transfected with the indicated plasmids. Cells were treated with 10 ug/ml cycloheximide for 5 h prior to harvest. After 48 h, proteins were separated by SDS-PAGE and analyzed by Western blotting using the specified antibodies. GAPDH is shown as an internal control. C. HEK293T cells were transfected with the indicated plasmids and after 48 h, IP was performed and analyzed as described in A. Unaltered blots are shown in Supplemental Fig S2.