HPat a Decapping Activator Interacting with the miRNA Effector Complex
Figure 3
Co-purification of HPat with GW182 in AGO1 knockdown cells.
A: Cells stably expressing HA-GW182 and Myc-HPat were treated for four days with dsRNA against YFP (control KD) or AGO1 (AGO1 KD). Protein complexes were immunoprecipiated from cell lysates using anti-HA antibody. Increasing amounts of the input sample and immunoprecipitates (IP) were analyzed by western blot analysis using anti-HA (Inputs lanes 1–4, 12–15, and IPs lanes 5 and 16) or anti-c-myc antibody (Inputs lanes 6–10, 17–21 and IPs lanes 11 and 22). The percentage of total cell lysate loaded in input lanes or the percentage of the total IP are indicated. B: Quantitative analysis of the western blot in (A). The amount of Myc-HPat/HA-GW182 in the IP was normalized and the value for the control IP set to 1. C: Knockdown efficiency of AGO1. Cell lysate of AGO1 knockdown cells and various amounts of control cell lysate were analyzed by western blot analysis. Tubulin was used as a loading control. D: Upregulation of endogenous miRNA targets CG5123 and CG6770 in AGO1 knockdown cells. Total RNA from input samples of (A) were analyzed by RT-qPCR and normalized to rp49 levels. The values of dsYFP treated cells were set to 1. In all figures bars represent mean values and error bars standard deviations of at least three biological replicates. Statistical analysis was performed using the Student’s t test and significance values are as follows: *, p<0.002; **, p<0.0001.