Figure 10. Activity of NOT1 protein domains in tethering assays. (A and B) A tethering assay was performed as described in Figure 9A with NOT1 (full-length or fragments). Firefly luciferase activity was normalized to that of Renilla luciferase and set to 100 in the cells expressing λN-HA. The mean values ± standard deviation from three independent experiments are shown in panel A. (B) northern blot of representative RNA samples analyzed as described in Figure 8B. (C and D) Tethering assay using the F-Luc reporter lacking the Box B hairpins. (E and F) Tethering assay using the unadenylated F-Luc-BoxB-HhR reporter. (G) western blot showing the expression level of the protein fragments tested. (H and I) The effect of the K1277A mutation (which disrupts POP2 binding)¹⁶ on the activity of NOT1-M and full-length NOT1 was tested in tethering assays using the F-Luc-5BoxB reporter as described above.