Fig 4. MDM2 E3 ligase activity is required p53-dependent cell cycle regulation in normal growth conditions.

(A) WB analysis of p53 expression (rabbit polyclonal antibody, Proteintech 10442-1-AP) in four (#1 to #4) E9.5 embryos of Mdm2^+/+: Trp53 ^R/+ (#1, #2) and Mdm2^la/+: Trp53 ^R/+ (#3, #4) status. Normalized p53 levels shown as fold/p53 after quantification of p53 and HSC70 (loading control) bands by ImageJ and normalized against HSC70. (B) WB analysis of p53, MDM2 and MDM4 protein expression levels in Mdm2^+/+-tetp53 and Mdm2^la/la-tetp53 MEFs. Doxycycline (Doxy, 200ng/ml) was administered for 1 day (1d) or 2 days (2d) with or without further treatment with proteasome inhibitor carfilzomib (CFZ, 400nM) for 8h. p53^-/-/Mdm2^-/-/Mdm4^-/- triple knockout MEFs (TripKO) were used as negative control. Normalized p53 levels against tubulin shown as fold/p53. (C) Left panel, Mdm2^+/+-tetp53 and Mdm2^la/la-tetp53 MEFs were treated with 200ng/ml doxycycline treatment for 24h followed by treatment with or without 400nM CFZ for 8h to assess p53 ubiquitination in cells. Denatured IP was performed with the cell lysates and p53 antibody (DO-1) followed by IB for polyubiquitin (left panel). Right panel, Direct IB for p53 and polyubiquitin and tubulin (loading control) with the same cell lysates are shown (right panel). (D-to-G) Cell cycle profiles of Mdm2^+/+-tetp53 MEFs (D & E) and Mdm2^la/la-tetp53 MEFs (F & G) at passage 10 in the absence (D, F) or presence (E, G) of p53 induction with 200ng/ml doxycycline treatment for 24h.