Fig. 4

S100A6 increases MDM2 self-ubiquitination by disrupting MDM2–HAUSP–DAXX interactions. A A schematic diagram showing different domains of MDM2 and its truncations. B GST pull-down assay for binding between GST-S100A6 and HA-MDM2 300–491, 300–436, and 437–491. C Co-immunoprecipitation assay for MDM2–HAUSP–DAXX interactions and binding between S100A6 and MDM2, HAUSP, or DAXX in MCF-7 cells in the presence of CaCl₂ or EGTA. D Expressions of HAUSP and DAXX in MCF-7 cells transfected with S100A6 (5 μg, S100A6 plasmid). E Expressions of HAUSP and DAXX in MCF-7 transfected with S100A6 siRNA (5 μg, siS100A6). Histograms showed the densitometric analyses of indicated proteins. Data were shown as mean ± SD; n = 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 F In vivo ubiquitination assay for testing the effect of HAUSP with or without S100A6 (2.5 and 5 μg, S100A6 plasmid) on MDM2 ubiquitination in MCF-7 cells. G CHX pulse-chase assay for testing the effect of HAUSP with or without S100A6 on MDM2 turnover. Curves for relative expression of MDM2 transfected with control plasmid (blue line), HA-HAUSP plasmid (red line), and HA-HAUSP and Flag-S100A6 plasmid (green line). Data were shown as mean ± SD; n = 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001