Figure 4A HEK293T cells were transfected with the indicated R-Luc-6xMS2-A95-MALAT1 reporters containing the indicated 5'-UTRs. An F-Luc reporter whose translation was dependent on the Hepatitis C virus (HCV) IRES was included as a control. One day after transfection, cells were treated with DMSO or silvestrol. R-Luc activity in silvestrol-treated cells was normalized to that measured in control cells. The corresponding values for the HCV IRES-F-Luc reporter are shown in Fig EV3H. The panel shows mean values ± standard deviations from three independent experiments. B-E Tethering assays using the indicated R-Luc-MS2-A95-MALAT1 reporters and MS2-HA-tagged TNRC6A-SD, AGO2 and NOT1 in HEK293T cells. R-Luc activity and mRNA levels were normalized to those of the F-Luc transfection control and set to 100% in cells expressing MS2-HA. Panels B,D show mean values ± standard deviations from three independent experiments. Panels C and E show Northern blots of representative RNA samples. B-E Tethering assays using the indicated R-Luc-MS2-A95-MALAT1 reporters and MS2-HA-tagged TNRC6A-SD, AGO2 and NOT1 in HEK293T cells. R-Luc activity and mRNA levels were normalized to those of the F-Luc transfection control and set to 100% in cells expressing MS2-HA. Panels B,D show mean values ± standard deviations from three independent experiments. Panels C and E show Northern blots of representative RNA samples. F-H Lysates from HEK293T cells expressing R-Luc-MS2-A95-MALAT1 reporter and MS2-HA tagged proteins were immunoprecipitated using anti-eIF4A2 antibody. The RNAs coimmunoprecipitating with eIF4A2 were analyzed by northern blot. 7SL RNA served as a loading control. R-Luc mRNA levels were normalized to those of the F-Luc control. The normalized values in the IP were divided by those in the input and set to 100 for cells expressing MS2 peptide. (F) Normalized R-Luc activities. The panel shows mean values ± standard deviations from five independent experiments. (G) Representative western and northern blots of input and IP fractions. For the northern blots blot, 2% of the input and 6% of the IP fraction were analyzed. For the northern blots 2% of the input and 40% of the IP were analyzed. (H) Efficacy of the immunoprecipitation. Error bars represent standard errors from five independent experiments. F-H Lysates from HEK293T cells expressing R-Luc-MS2-A95-MALAT1 reporter and MS2-HA tagged proteins were immunoprecipitated using anti-eIF4A2 antibody. The RNAs coimmunoprecipitating with eIF4A2 were analyzed by northern blot. 7SL RNA served as a loading control. R-Luc mRNA levels were normalized to those of the F-Luc control. The normalized values in the IP were divided by those in the input and set to 100 for cells expressing MS2 peptide. (F) Normalized R-Luc activities. The panel shows mean values ± standard deviations from five independent experiments. (G) Representative western and northern blots of input and IP fractions. For the western blot, 2% of the input and 6% of the IP fraction were analyzed. For the northern blots 2% of the input and 40% of the IP were analyzed. (H) Efficacy of the immunoprecipitation. Error bars represent standard errors from five independent experiments.