Fig. 5. MAE localization in S2 cells depends on the distribution of its binding partners. (A,B) Immunoblots of MYC-IPs visualized with anti-MYC (MAE), anti-YAN and anti-FLAG (PNT-P2). MAE complexes with YAN in the absence of RAS/MAPK signaling (A) and with PNT in the both the absence and presence of signaling (B). Lanes are from the same gel and immunoblot, but have been rearranged. Lanes 1, 3, 5 are non-IPed lysates; lanes 2, 4, 6 are the corresponding IPs. Specificity of the anti-MYC IP is demonstrated in lane 6 of A and B, where in the absence of MAE, YAN or PNT-P2 are not precipitated. (C-O) Anti-MYC staining of S2 cells transfected with MYC-mae. (C'-O') DAPI staining of the same cells. (C,E,G,I,K,M) Absence of RAS^V12. (D,F,H,J,L,N,O) Presence of RAS^V12. For each experiment (C-O), the percentage of transfected cells exhibiting nuclear localization (G,K-O), or both nuclear and cytoplasmic localization (C-F,H-J) is indicated. n, number of cells scored in each experiment. (C,D) MAE; (E,F) MAE+RNAi crm1; (G,H) MAE+YAN; (I,J) MAE+YAN^N'; (K,L) MAE+YAN^ACT; (M,N) MAE+PNT-P2; (O) MAE+YAN+RNAi crm1. MAE is ubiquitously expressed in S2 cells (C,D), except when YAN or PNT-P2 is co-transfected. When MAE is co-transfected with wild-type YAN, MAE is nuclear in the absence of signaling (G) and becomes nuclear and cytoplasmic in the presence of RAS^V12 (H). CRM1 does not mediate the export of MAE (E,F). However, when YAN is co-transfected with MAE, and CRM1-mediated export is inhibited by RNAi, MAE remains nuclear (O). MAE interacts with YAN via the PD, as MAE is ubiquitously expressed when YAN^N' is co-expressed (I,J). Co-transfection of MAE with YAN^ACT restricts MAE to the nucleus in the absence and presence of RAS^V12 (K,L). Similarly, PNT-P2 restricts MAE to the nucleus (M,N).

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