Figure 4

HIPK2-mediated β-catenin proteasomal downregulation independent of p53 and GSK3β activities. H1299 (p53/null) lung adenocarcinoma cells were transfected with HA-β-catenin and Flag-HIPK2 expression vectors, in the presence or absence of the proteasome inhibitor MG132 (2.5 μM) for 6 h, or the specific GSK3β inhibitor LiCl (30 mM for 16 h). Thirty-six h after transfection, total cell extracts were analysed by Western immunoblotting with anti-HA (to detect β-catenin), anti-phospho-GSK3 (to detect Ser21 and Ser9 phosphorylations of respectively GSK3α and GSK3β), and anti-GSK3 (to detect total GSK3α and GSK3β proteins) antibodies. Anti-tubulin was used as protein loading control.