Fig. 3. CRM1-mediated nuclear export of YAN requires both the NESs and the PD. (A-M) S2 cultured cells transfected with various YAN deletion constructs and stained with anti-YAN. (A'-M') DAPI staining of the same cells. For each experiment (A-M), the percentage of transfected cells exhibiting nuclear localization (A,C,D,F,G-I,L,M), both nuclear and cytoplasmic localization (J,K), or exclusively cytoplasmic localization (B,E) is indicated. n, number of cells scored in each experiment. (A-B,D-E) Deletion of the first or first and second NES has no effect on export. (G-H,J-K) Deletion of the third NES and majority of the PD results in inappropriate export in the absence of signaling, and impairs export in the presence of RAS^V12. (L-M) Deletion of the whole N terminus completely inhibits export. (C,F,I) RNAi-mediated knockdown of crm1 restricts YAN to the nucleus in the presence of RAS^V12. (O) Transcription assays with YAN^N' and YAN^N3+PD show that both deletions repress transcription and are responsive to RAS^V12.