FIGURE 4.
PCNA is dispensable for the degradation of Gcn5 by CRL4Cdt2 E3 ligase. A, Gcn5 does not interact with PCNA. Left, 293T cells transfected with the indicated plasmids were harvested for immunoprecipitation (IP) using anti-FLAG M2-agarose beads. FLAG precipitates were subjected for immunoblot using anti-FLAG, anti-And-1, or anti-PCNA antibody. Note that And-1 was detected in FLAG-Gcn5 precipitates. Right, 293T cells were harvested for immunoprecipitation using anti-Gcn5 antibody or IgG. The Gcn5 precipitates were subjected for immunoblot using anti-And-1, anti-Gcn5, or anti-PCNA antibody. B, down-regulation of PCNA does not stabilize Gcn5 proteins in cells with depleted And-1 by siRNA. Left, U2OS cells transfected with the indicated siRNAs were harvested and subjected to immunoblot using anti-Gcn5, anti-PCNA, anti-And-1, or anti-actin. Right, quantification of Gcn5 protein levels. Means ± S.D. (error bars) were obtained from four independent experiments. The relative levels of Gcn5 proteins were measured as in Fig. 1B.