Fig. 2

The coiled-coil region of the N-terminus of PSMC3 interacts with human AGO2. a PSMC3 interacts with AGO2 in an RNA-independent manner. HeLa cells were co-transfected with HA-AGO2-PIWI and Flag-PSMC3. Immunoprecipitation assays were performed with anti-HA antibody and western blotting with anti-Flag and anti-HA antibodies. For lanes 4, 5, and 6, cell lysates were incubated at room temperature with RNase A (10 mg/ml) for 30 min. b PSMC3 colocalizes with AGO2 in HeLa cells. Myc-PSMC3 was transfected into HeLa cells. Forty-eight hours after transfection, immunofluorescence assays were performed with the indicated antibodies, then imaged by confocal microscopy. Scale bar, 10 µm. c Western blotting assays were used to detect AGO2 and PSMC3 protein levels in nucleoplasm or cytoplasm from HeLa cells. d PSMC3 interacts with AGO2 in both nucleoplasm and cytoplasm. HeLa cells were co-transfected with HA-AGO2 -PIWI and Flag-PSMC3. Then nucleoplasmic or cytoplasmic extracts were harvested and analyzed by western blotting assays. e Schematic representation of the full-length and truncated PSMC3s used to map AGO2-binding sites. PSMC3 possesses an N-terminal domain and an AAA ATPase domain that contains an ATP-binding motif (black box) and a helicase motif (white box). f A yeast two-hybrid system was used to determine whether pSOS-AGO2-PIWI could interact with the full-length PSMC3 protein or with deletion mutants PSMC3 (214–439), PSMC3 (354–439), PSMC3 (1–214), and PSMC3 (97–439). g Mapping the AGO2-interacting site in PSMC3. HeLa cells were transfected with the indicated plasmids encoding Flag-tagged full-length PSMC3 or PSMC3 deletion mutants. After 48 h, cell lysates were immunoprecipitated with anti-AGO2 antibody and then western blotting was performed with anti-Flag and anti-AGO2 antibodies. All results are representative of three independent experiments