Fig. 4

PSMC3 is essential to maintain AGO2 protein levels. a PSMC3 increases AGO2 protein levels. HeLa cells were transfected with the indicated plasmids. Extracts were collected at 48 h post-transfection and subjected to western blotting analysis with anti-AGO2 and anti-GAPDH antibodies. b Expression of siRNA-resistant PSMC3 abrogates the suppression of AGO2 caused by PSMC3-specific depletion. HeLa cells were co-transfected with plasmids encoding either the wild-type (wt) or mutant (mut) PSMC3 along with siPSMC3 or control siRNA. After 48 h, lysates were analyzed by western blotting with anti-AGO2 and anti-GAPDH antibodies. c Real-time RT-PCR analysis of endogenous AGO2 mRNA was performed using total RNA isolated from HeLa cells after 48 h of transfection with the indicated plasmids. GAPDH mRNAs served as the control. In all statistical comparisons, three independent experiments were performed (mean ± SD, n = 3, Student’s t-test). d Depletion of PSMC3 increases AGO2 protein turnover. HeLa cells were transfected with control or PSMC3 siRNA. After 48 h, the cells were treated with 100 µg/mL of cycloheximide (CHX) for the indicated periods and then harvested for immunoblotting with anti-AGO2 and anti-GAPDH antibodies (left). The results were plotted after quantitation (right). e Depletion of PSMC3 decreases the amount of AGO2 in the cytoplasm. HeLa cells were co-transfected with the indicated plasmids. Then nucleoplasmic or cytoplasmic extracts were harvested and analyzed by western blotting assays with anti-Flag, anti-GAPDH (cytoplasmic marker), and anti-LMNB1 (nucleoplasmic marker) antibodies. All results are representative of three independent experiments