Lis1 promotes the recruitment of dynein and dynactin to RNAs. (A) Immunoblots showing that the recruitment of Dhc, but not Egl and BicD, to the ILS is dramatically reduced when complexes are assembled from lis1 mutant ovary extract using aptamer-based RNA pull-downs. Recruitment of proteins to the ILS^as mutant from WT extract is shown as a specificity control. Note that reduced Lis1 levels in the starting extract are not apparent for the heterozygous lis1^E415/lis1⁺ combination due to nonlinear enzyme-coupled chemiluminescent detection (reduction of Lis1 levels in heterozygotes is revealed by nonenzymatic detection with fluorescent antibodies [Figs. 4 A, 7 C, and Fig. S4 D]). (B) Immunoblots showing a reduced association of p150^Glued and p50^Dmn with the aptamer-associated ILS when complexes are assembled from lis1 mutant ovary extract compared with WT. Blots for Egl and Dhc recruitment to signals are shown as controls. (C) Immunoblots assessing protein recruitment from WT and lis1 mutant ovary extracts to a localizing 800-nt piece of the K10 3′UTR (K10^3′UTR) and an antisense nonlocalizing mutant version (asK10^3′UTR). Reduced Lis1 levels have no effect on Egl binding, but clearly decrease the association of Dhc with both RNA species. We confirmed that the association of p150^Glued with asK10^3′UTR was also reduced using the lis1 mutant versus WT extract (Fig. S3 D). (D) Cartoon summarizing the results from A–C and Fig. S3 D. Localizing RNAs recruit dynein–dynactin complexes to sites on localization signals bound by Egl–BicD complexes, and to additional sites on the RNA bound via an unknown protein(s) (factor X), also present on nonlocalizing RNAs (see also Bullock et al., 2006). Reduced Lis1 levels decrease the affinity of dynein and dynactin complexes for both signals and nonsignal sites.