Fig. 3

Effects of S100A6 on MDM2 self-ubiquitination and protein degradation. A Turnover of MDM2 in MCF-7 cells under transfection of S100A6 (5 μg, S100A6 plasmid) and control plasmid (vehicle) by CHX pulse-chase assay. Curves for relative expression of MDM2 of transfection of S100A6 (red line) and control plasmid (blue line) were shown. Data were shown as mean ± SD; n = 3 independent experiments. ***P < 0.001. B The effect of S100A6 on MDM2 ubiquitination in MCF-7 cells under transfections of various concentrations of S100A6 in an in vivo ubiquitination assay. C The effect of S100A6 (0, 2.5, and 5 μg, S100A6 plasmid) on the ubiquitination of full-length MDM2 or its fragments 1–299 and 300–491 in an in vivo ubiquitination assay. D The effect of GST-S100A6 on the ubiquitination of HA-tagged full-length MDM2 or its fragments 1–299 and 300–491 in an in vitro ubiquitination assay. E Sequence of the C-terminal RING domain of MDM2 and its mutants C461S, C464A, and C478S. F The effect of S100A6 (5 μg, S100A6 plasmid) on the ubiquitination of either wild-type MDM2 or mutant MDM2 C461S, C464A, and C478S in an in vivo ubiquitination assay. G The effect of GST-S100A6 on the ubiquitination of HA-MDM2 C461S, C464A, and C478S in an in vitro ubiquitination assay