Fig_1.tif (3.26 MB)

Glis2 interacts with PIAS4 and SUMO-3.

figure

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

(A) FLAG-tagged PIAS4 (F.PIAS4) and V5-tagged Glis2 (V5.Glis2) were transiently co-expressed in HEK 293T cells together with F.CD2AP and V5.Diversin as controls. Precipitation of F.PIAS4 immobilized only V5.Glis2 but not V5.Diversin, while V5.Glis2 was not detectable in F.CD2AP precipitates. (B) Immunofluorescence analysis of HEK 293T cells expressing YFP.PIAS4 (green) and RFP.Glis2 (red) showed nuclear co-localisation of both proteins. Nuclei were stained with Hoechst blue. (C) His-tagged SUMO proteins (SUMO-1, SUMO-2, and 3) were transiently co-transfected with V5.Glis2, V5.NPHP3 and V5.NPHP2 in HEK 293T cells. After 36 hours of transfection, SUMO proteins were precipitated using Ni-NTA beads. Glis2 and other nephronophthisis gene products (NPHP) were detected using an anti-V5 antibody.