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. 2016 Apr 25;113(19):5263–5268. doi: 10.1073/pnas.1524291113

Fig. S3.

Fig. S3.

Effects of importins on the nuclear export activity of eIF4E. (A) Importin 8 knockdown (shImp8) in cells with endogenous levels of eIF4E (FLAG vector cells) reduces the mRNA export of eIF4E target mRNAs, but not negative controls (Top), with no changes to total mRNA levels (Middle). (Bottom) Cytoplasmic-to-nuclear ratio (c/n) of mRNA was obtained using fractionation and qPCR for the indicated mRNAs. For fractionation controls, we monitored U6snRNA for the nucleus, and tRNA lysine for the cytoplasm. N and C indicate nuclear and cytoplasmic fractions, respectively. (B) Consistently, importin 8 knockdown leads to reduced protein levels of eIF4E mRNA export targets but not eIF4E or negative controls, such as survivin and β-actin. (C) Fractionation controls for mRNA export assays in Fig. 2. (D) Total mRNA levels (Fig. 2) did not change upon knockdown or overexpression of importin 8. (E) mRNA export assays (Top) and total mRNA levels (Bottom) on indicated RNAs upon siRNA knockdown of importin 8 or importin 9 in the presence of overexpressed eIF4E (FLAG eIF4E). (F) Western blot analysis indicating eIF4E export targets were reduced by importin 8 knockdown in eIF4E-overexpressing cells but not by importin 9 compared with control knockdowns in the presence of eIF4E overexpression. (G and H) As in E and F, but in the vector control (FLAG vect) cells, where the effects on endogenous eIF4E can be monitored. Similarly, knockdown of importin 8 affects mRNA export and protein production of targets, but not importin 9. In E–H , it is demonstrated that importin 8 modulates the effects of both exogenous and endogenous eIF4E. (I) Fractionation controls for E and G. U6snRNA is a marker for the nucleus, and tRNA lysine is a marker for the cytoplasm. (J) Western blot demonstrating that importin 9 overexpression does not increase levels of eIF4E target RNAs, consistent with its inability to bind or import eIF4E (Fig. S1 B and D). (K) Fractionation analysis of importin 8-overexpressing cells demonstrating that eIF4E is more nuclear upon importin 8 overexpression relative to vector or importin 9-overexpressing cells. α-Tubulin and MEK-1 are fractionation controls for the cytoplasm, and Sc35 and Pol II are fractionation controls for the nucleus.