Fig. 4

Icaritin stabilized p53 by inhibiting Mdm2-mediated p53 ubiquitination. a Icaritin decreased expression of Mdm2. Mdm2 and GAPDH (as a control) was detected by western blot after DMSO or 20 μM icaritin treatment in HepG2 cells and SMMC7721 cells. b Icaritin inhibited interaction of protein Mdm2 and p53. IP assay was carried out by using p53 antibody and followed by western blot with p53 or Mdm2 antibody (top). After DMSO or icaritin treatment, IP assay was carried out by using p53 antibody and followed by western blot with p53 or Mdm2 antibody. Samples were immunoblotted against p53 to determine immunoprecipitation efficiency (lower right). c Overexpression of Mdm2 rescued p53 ubiquitination in HepG2 and SMMC7721 cells. HepG2 and SMMC7721 cells were cotransfected with vector FLAG, FLAG-Mdm2, HA-ubiquitin and then DMSO or icaritin treatment. P53 protein was then isolated by immunoprecipation and Ub-p53 was detected by western blot using anti-ub antibody. P53 and Mdm2 were detected by western blot in cells cotransfected with FLAG, FLAG+icaritin, FLAG-Mdm2 + icaritin. The full-length images for gels/blots in Fig. 4a, b and c were presented in Supplementary Fig. 7