Figure 3

Sumoylation inhibits HOXA10 protein stability and transcriptional activity. (a) Ishikawa cells were transfected with Myc-HOXA10^WT or Myc-HOXA10^K164R and Flag-SUMO1. After 48 h, 50 μg/ml CHX was added to the cells for 2, 4 and 8 h. The cell extracts were subjected to western blot analysis. The levels of remaining HOXA10 were normalized to GAPDH and plotted relative to the levels at the 0-h time point. Values are expressed as the means±S.E.M. (n=3), *P<0.05 versus HOXA10^WT by itself. (b) For detection of ubiquitination. Ishikawa cells were treated with 10 μM MG132 for 6 h before being harvested and subjected to immunoprecipitation with anti-Myc antibody, followed by western blot analysis to assess protein expression. (c) Ishikawa cells were transfected with ITGB3-Luc, Myc-HOXA10^WT, Myc-HOXA10^K164R and Flag-SUMO1 as indicated. After 48 h, the luciferase activities were measured, and the activity levels are presented as the fold induction. The error bars indicate±S.E.M. of three independent experiments. **P<0.01; ***P<0.001. (d) For the biotin-labeled DNA pull-down assay, Ishikawa cell extracts were incubated with biotinylated DNA probe containing HOXA10-binding sites (TTAT). After the cell extracts were incubated with streptavidin Sepharose beads, the extracts were subjected to western blot analysis.