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. 2014 Nov 21;3(12):1207–1216. doi: 10.1242/bio.20149308

Fig. 6. NANOS3 associates with the CCR4-NOT deadenylation complex via a direct interaction with CNOT8.

Fig. 6.

(A–C) GST pull-down assay. Protein extracts from E. coli expressing GST-fused CNOT1-1, 1-2, 1-3, 2, 3, 4, 6, 6L, 7, 8, 9, 10, or 11/D1Bwg0212e were combined with MBP-tagged NANOS2 or NANOS3 (A). GST-fused CNOT1-1, 1-2, or 1-3 were expressed in E coli as described previously (Suzuki et al., 2012). Protein extracts from these E. coli. were combined with MBP-NANOS2 or NANOS3 (B). Protein extracts from E. coli expressing GST-fused CNOT8 were combined with MBP-tagged NANOS2 or NANOS3 (C). CNOT proteins precipitated with glutathione sepharose were visualized by staining with Coomassie brilliant blue, whereas co-precipitated MBP-NANOS2 or NANOS3 were detected by western blotting. Red arrowheads in (B) and (C) indicate MBP-NANOS3 co-precipitated with CNOT8. (D) Flag-tagged NANOS2 or NANOS3 were immunoprecipitated and then incubated with a 5′-fluorescein isothiocyanate-labeled poly(A) RNA substrate for 0, 45, 90, and 180 minutes. Samples were then analyzed on a denaturing sequencing gel. (E) Flag-tagged NANOS2 or NANOS3 were transfected into HeLa cells and then precipitated with anti-FLAG antibodies from protein extracts. Precipitates were analyzed by western blotting with the indicated antibodies.