Figure 3
PIAS1 promotes FOXP2 SUMOylation.
(a) Gel shift assay for SUMOylation of FOXP2. HEK293 cells were transfected with V5-tagged FOXP2 and mCherry-SUMO, together with myc-tagged PIAS1 (+) or an empty vector (−). Top panel: western blot probed with anti-V5 antibody. The 90 kDa species is unmodified FOXP2. The 140 kDa species is FOXP2 modified with mCherry-SUMO. Second panel: western blot probed with anti-mCherry. The asterisk indicates unconjugated mCherry-SUMO. Higher molecular weight species are cellular proteins modified with mCherry-SUMO. Third panel: western blot probed with anti-myc tag to detect PIAS1. Bottom panel: western blot probed with anti-β-actin to confirm equal loading. (b) Top: schematic representation of PIAS1 C350S mutant. Bottom: BRET assay for interaction of FOXP2 with PIAS1. HEK293 cells were transfected with luciferase-FOXP2 (donor) and YFP-PIAS1 (wild-type (WT) or C350S mutant, acceptor). The control donor protein is a nuclear-targeted luciferase. Values are mean corrected BRET ratios ± S.E.M. (n = 3). (c) Fluorescence micrographs of HEK293 cells transfected with mCherry-tagged wild-type PIAS1 (WT) or C350S mutant (red) and YFP-FOXP2 (green). Nuclei were stained with Hoechst 33342 (blue). (d) Gel shift assay for SUMOylation of FOXP2. HEK293 cells were transfected with FOXP2-UBC9 together with mCherry-SUMO or mCherry alone (control) and myc-tagged wild-type PIAS1 (+), C350S mutant (M) or empty vector (−). Top panel: western blot probed with anti-V5 antibody to detect FOXP2-UBC9. The 110 kDa species is unmodified FOXP2-UBC9. The 130 kDa species is FOXP2-UBC9 modified with endogenous SUMO. The 170 kDa species is FOXP2-UBC9 modified with mCherry-SUMO. Second panel: western blot probed with anti-mCherry antibody. The asterisk indicates unconjugated mCherry-SUMO. Higher molecular weight species are cellular proteins modified with mCherry-SUMO. Third panel: western blot probed with anti-myc to detect PIAS1. Fourth panel: western blot probed with anti-β-actin to confirm equal loading. Bottom panel: densitometry analysis of FOXP2-UBC9 species.
