FIG. 4. Suppression of siRNA-based RNAi by ADAR1.
A, effects of endogenous ADAR1 proteins. A combination of varying concentrations of 19-bp EGFP siRNA and nonspecific control siRNA together with pEGFP-C1 target (1.5 µg) and pCMV-DsRed-Express control (1.5 µg) plasmid DNA were co-transfected into wild-type, PKR null, ADAR2 null, and ADAR1 null MEF cells. Quantitative measurement of EGFP and RFP expression levels were done by FACS analysis, and the EGFP expression level relative to that of RFP was determined. Results are presented as means ± S.E. (bars) values of five independent experiments performed in triplicate. The reduction in the relative EGFP expression levels in ADAR1 null MEF cells was examined statistically by individual unpaired Student’s t tests in comparison to those in wild-type, PKR null, and ADAR2 null MEF cells. p < 0.001 for both 0.5 and 1 nm concentrations. B, effects of recombinant ADAR1p150 proteins. In addition to pEGFP-C1 and pCMV-DsRed-Express, 3.0 µg of pCMV-F-ADAR1p150 or pCMV-F-ADAR1ΔM1M2M3 were transfected into ADAR1 null MEF cells. The vector-only control experiments were also conducted with pRC/CMV plasmid DNA. Results are presented as the mean ± S.E. (bars) values from three independent experiments performed in triplicate. The difference between the vector-only control or pCMV-F-ADAR1ΔMIM2M3 experiments and the pCMV-F-ADAR1p150 experiments (0.5, 1.0, and 2.0 nm concentrations) were significant (p < 0.001) by unpaired Student’s t tests. C, the total protein extracted from 1 × 105 ADAR1 null MEF cells transfected for 44 h with vector-only (lane 3), pCMV-F-ADAR1p150 (lane 4), or pCMV-F-ADAR1ΔMIM2M3 (lane 5), together with varying amounts of FLAG epitope-tagged p150 standard proteins (0.5 or 2 fmol) was examined by Western blotting analysis.