Figure 4.

RASSF1A interacts with MDM2 in vivo and in vitro. (A) Lysates from p53^+/+, p53^−/− and Mdm2^−/−p53^−/− MEFs were immunoprecipitated with anti-Mdm2 antibodies and the resulting immunoprecipitates were analysed by immunoblotting. Asterisk indicates nonspecific bands. (B, C) The indicated fragments of RASSF1A and MDM2 were produced by in vitro translation in the presence of [³⁵S]methionine. Fragments were mixed, immunoprecipitated with anti-Flag (B) or anti-HA (C) antibodies and analysed by SDS–PAGE and autoradiography. (D) Self-ubiquitylation of GST–MDM2 and GST–MDM2(ΔRING) with or without GST–RASSF1A was detected using ³²P ubiquitin or immunoblot analysis (IB) with α-GST. Molecular weights are in kDa.