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. 2004 Sep;24(17):7654–7668. doi: 10.1128/MCB.24.17.7654-7668.2004

FIG. 2.

FIG. 2.

L23 interacts with MDM2 in cells. (A) Exogenous MDM2 and L23 interact with each other in 293 cells. HA-MDM2 (1.5 μg), Flag-L23 (1.5 μg), or both vectors (1.5 μg each) were used for transfection, as indicated at the top. Whole-cell lysates (500 μg) were subjected to immunoprecipitation (IP) with anti-HA (α-HA) or a control antibody followed by immunoblotting (IB) with anti-Flag (α-Flag) (upper) or anti-HA (lower) antibodies. IgG, immunoglobulin G. (B) The same transfections as shown in panel A were conducted except that anti-Flag antibodies were used for immunoprecipitation. (C) MDM2 colocalized with L23 in both the nucleus and the cytoplasm but not in the nucleolus. 293-HA-MDM2 cells were transfected with Flag-L23 and immunostained with both polyclonal anti-MDM2 (red) and monoclonal anti-Flag (green) antibodies. (D) L23 binds to MDM2 deletion mutants in cells. 293 cells were transfected with 6 μg of wild-type MDM2, MDM2Δ150-230, or empty (−) plasmids, as indicated at the top. Whole-cell lysates (500 μg) were immunoprecipitated with anti-L23 (α-L23) antibodies followed by immunoblotting with anti-MDM2 (2A10) and anti-L23 antibodies. Ten percent of the lysates loaded as input are shown in the panels on the right side. (E) Endogenous L23 interacts with endogenous MDM2 in U2OS cells. Whole-cell lysate (500 μg) was used for immunoprecipitation with either rabbit polyclonal anti-L23 antibody or preimmune serum (control), followed by immunoblotting with anti-MDM2 (2A10) (top panel) or anti-L23 (bottom panel) antibody.