AGO2 interacts with TP53 in an RNA-dependent manner. HCT116 TP53+/+ were treated with vehicle or DOX for 12 h, and coimmunoprecipitation assays were then performed to determine whether an interaction exists between TP53 and AGO2 proteins. (A) Reciprocal co-IP experiments using AGO2 and TP53 antibodies demonstrate an interaction between the two proteins. DDX5 was used here as a positive control for the immunoprecipitation, and ACTB was used as a loading control housekeeping gene in the input sample. (B) To test the impact of mutations in TP53 on its ability to interact with AGO2, TP53−/− cells were transfected with empty plasmid (null) or plasmids expressing mutated forms of TP53 (R175H, R248W, and R273H), treated with DOX for 12 h, and subjected to co-IP. (C) To investigate whether the association between TP53 and AGO2 is RNA-dependent, immunoprecipitates of AGO2 and TP53 from cells treated with DOX for 12 h were digested with increasing quantity of RNase A for 30 min at 37°C, separated into supernatant and beads, and analyzed by immunoblotting. At least three independent experiments have been performed in all cases.
