Figure 1

Effect of homodimerization on MDM2 protein stability. A, homodimerization by DSS (0.5 mM) cross-linking for 30 min, with subsequent expression of proteins as indicated, in their monomer and homodimer forms in NB-1691 and LA1-55N cells were detected by Western blot assays. B, dose–response expression of MDM2 and MDM4 proteins after treatment with increasing concentrations (0.25, 0.5 and 1 mM) of DSS. C, homodimerization by DSS and expression of transfected HA-tagged full-length MDM2 and various mutated or deleted MDM2 fragments in SK-N-SH cells, as detected by Western blot using anti-HA (tagged) antibody. D, SK-N-SH cells were co-transfected with consistent amounts (3 μg) of Myc-tagged full-length MDM2 (Myc-MDM2/1–491) and increasing amounts (1, 2 and 3 μg) of either HA-tagged wt-RING domain of MDM2 (HA-MDM2/415–491) or 464 mutated (464 m) RING domain of MDM2. The expression of transfected full-length MDM2 and the RING domains of MDM2 was detected by Western blot, using anti-Myc and anti-HA antibodies, respectively. E, turnover of transfected MDM2 (as detected by anti-Myc antibody) in SK-N-SH cells, in the presence or absence of HA-MDM2/415–491, as detected by pulse-chase assay. Numerical labels under each MDM2 band represent their relative expression levels after normalization for GAPDH, as compared with untreated (0) samples that were defined as 1 unit. F, a similar co-transfection and Western blot assay as described in (D) for the Myc-tagged full-length MDM2 and the HA-tagged RING domain of MDM4 (421–490).