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. 2004 Dec 2;24(1):160–169. doi: 10.1038/sj.emboj.7600486

Figure 6.

Figure 6

Effects of Mdm2 siRNA on ubiquitination of pRB. (A) Knockdown of Mdm2 results in accumulation of pRB. U2OS cells were multiply transfected with 40 nM siRNAs targeting human Mdm2 (Mdm2 siRNA) or control oligo RNA (control siRNA) as described in Materials and methods. Cell lysates were subjected to IB with antibodies as indicated. Quantitative data are represented as fold values of control siRNA and means±s.e. of 3–4 separate experiments. (B) Knockdown of Mdm2 interferes with ubiquitination of pRB. In vivo ubiquitination of pRB in U2OS cells, in which Mdm2 is downregulated by RNAi, was performed. At 24 h after the third transfection of siRNA, the expression plasmids of pRB and HA-Ub were transfected into the cells. The levels of Mdm2 in siRNA-treated cells were analyzed by IB with anti-Mdm2 or anti-α-tubulin antibody (left panel). Double IP with anti-pRB antibody (αRB) or control immunoglobulin (cont-IgG) was performed as described in Materials and methods. The precipitates were then subjected to IB with anti-HA antibody for the detection of ubiquitinated pRB (right panel).