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. 2012 Jul 12;287(35):29373–29383. doi: 10.1074/jbc.M112.354001

FIGURE 5.

FIGURE 5.

The 7SL RNA affects the activity of A3G to counteract miRNA-mediated gene silencing. A, 293T cells were co-transfected with A3G-FLAG- (or A3G-W127L-FLAG-), MOV10-FLAG- and HA-AGO2-expressing plasmids. After 24 h, cells were lysed and co-immunoprecipitated with anti-HA agarose beads. The immunoprecipitated samples were then detected by Western blotting using anti-FLAG and anti-HA antibody. B, 293T cells were co-transfected with pcDNA3.1-A3G-HA (or pcDNA3.1-A3G-W127L-HA) and a plasmid containing the Renilla luciferase reporter gene with binding sites for mir25 in the 3′-UTR (the plasmid also contains the firefly luciferase gene as an intraplasmid control). pcDNA3.1 and psiCHECK-2 were also transfected as controls. At 48 h post-transfection, Renilla/firefly luciferase activity was measured. C, 293T cells expressing control-shRNA or SRP14-shRNA were first transfected with SRP14 expressing plasmid (pcDNA3.1 as a control). After 48 h post-transfection, 7SL RNA in transfected-cells was quantified by real-time RT-PCR. The level of 7SL RNA in cells expressing control-shRNA and pcDNA3.1 was defined as 100%. D, 293T cells expressing control-shRNA and those expressing SRP14-shRNA were first transfected with SRP14 expressing plasmid. At 24 h post-transfection, the transfected cells were then co-transfected with MOV10-FLAG- and A3G-HA- (or A3G-W127L-HA-) expressing plasmids. After 24 h, cells were lysed and co-immunoprecipitated with anti-HA-agarose beads. The precipitated samples were analyzed by immunoblotting using anti-FLAG or anti-HA antibody. E, 293T cells expressing control-shRNA or SRP14-shRNA were first transfected with SRP14 expressing plasmid. After 12 h, the transfected cells were then further transfected with A3G-expressing plasmid and a plasmid containing the Renilla reporter gene with binding sites for mir25 in the 3′-UTR (the plasmid also contains the firefly luciferase gene as an intraplasmid control). At 36 h post-transfection, the cells were harvested and disrupted, and Renilla/firefly luciferase activity was measured. Values in A and D represent portion of MOV10-FLAG normalized against HA-AGO2 (or A3G-HA) and compared with control. Data in B, C, and E represent mean ± S.D. (error bars). **, statistically significant, ≤0.01.