FIG 5.

14-3-3 proteins block FbxO11 from binding and ubiquitinating Cdt2. (A) 293T cells were transfected with plasmids as indicated. The procedure was otherwise as described in the legend to Fig. 4B. The bottom bands represent 0.5% of the input lysates. (B) 293T cells were treated with the indicated control GL2, 14-3-3γ, or 14-3-3ε siRNAs for 48 h. Before harvesting, cells were treated with MG132 (40 μM) for 6 h to allow the accumulation of Cdt2. The lysates were immunoprecipitated with anti-Cdt2 antibodies and probed with anti-Cdt2 and anti-FbxO11 antibodies (bottom). Top, input lysates. (C) Coimmunoprecipitation of PCNA and Cdt2. 293T cells were treated with the siRNAs indicated and MG132 as described for panel B. The extracts were immunoprecipitated with anti-PCNA antibodies and probed with anti-Cdt2 and anti-PCNA antibodies (bottom). Top, input lysates.