Fig_2.tif (2.55 MB)

Glis2 is SUMOylated by PIAS4/SUMO-3.

figure

posted on 2015-06-17, 04:52 authored by Haribaskar Ramachandran, Konstantin Herfurth, Rudolf Grosschedl, Tobias Schäfer, Gerd Walz

(A) HEK 293T cells were transfected with the plasmids as indicated. After 36 hours of transfection, whole cell lysates were prepared under denaturing conditions, and resolved on SDS-PAGE. Following Western blotting, Glis2 was detected using an anti-V5 antibody, YFP.PIAS4 using an anti-GFP antibody, and SUMO-3 using an anti-SUMO-3 antibody. SUMOylated Glis2 species are marked as SUMO-Glis2. (B) In vivo SUMOylation assay was performed to compare the SUMOylation of Glis2 and NPHP5 in the presence of SUMO-3 and PIAS4. Only SUMOylated Glis2 species, but no SUMOylated NPHP5 was detectable. (C) Co-transfection of FLAG-tagged PIAS4 (F.PIAS4) but not F.PIAS1 promoted the SUMOylation of Glis2 in the presence of SUMO-3. (D) V5.Glis2 was co-transfected with F.PIAS4 wild type (F.PIAS4.WT) and PIAS4, lacking E3 SUMO ligase activity (F.PIAS4.LD) together with His.SUMO-3. SUMOylated Glis2 was detected only with the wild type but not with the E3 ligase-deficient mutant of PIAS4.