Supplementary Figure 3: Exportin-5 interacts with ARF6, affecting TMV cargo.

a. PRISM predicted interaction between Exportin-5 and ARF6–GDP. b. Myc immunoprecipitation from cells expressing ARF6-WT-HA and myc-Exportin-5. Representative blots (N=3 biologically independent experiments) shown. c. Myc-tag co-immunoprecipitation from cells expressing ARF6-T27N-HA or ARF6-WT-HA; and myc-Exportin-5. Representative blots (N=3 biologically independent experiments) shown. Endogenous Exportin-5 and ARF6 imaging in LOX cell TMVs (d) and perinuclear region (e). Panels d and e represent orthogonal view of Fig. 3h. Representative images (N=3 biologically independent experiments) shown. f. Immunofluorescence of endogenous Exportin-5 and ARF6 in isolated TMVs. Representative images shown (N=4 biologically independent samples). g. Western blot of Exportin-5 in TMVs from invasive tumour lines. Representative blots (N=4 biologically independent experiments) shown. h. Dicer and Argonaute-2 western blotting in control or LOX^ARF6-Q67L TMVs. Representative blots (N=3 biologically independent samples) shown. i. qRT–PCR of TMV miRNA cargo from control or TBB treated cells. Data presented as mean±SD of N=3 biologically independent experiments. p-values determined by unpaired, two-tailed t-test between control and treatment reactions for each miRNA. j. Western blot of Exportin-5 in 20 μg total protein from control or TBB treated LOX cells. Representative blots (N=3 biologically independent samples) shown. k. Western blot of equal amounts of nuclear and cytosolic fractions from control or TBB treated LOX cells. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test between each control and treatment condition. l. Western blot of Exportin-5 in 20 μg total protein from control or TBB treated LOX^ARF6-Q67L cells. Representative blots (N=3 biologically independent samples) shown. m. Western blot of equal amounts of nuclear and cytosolic fractions from control or TBB treated LOX^ARF6-Q67L cells. Representative blots (N=3 biologically independent samples) shown. Data presented as mean±SD. p-values determined by unpaired, two-tailed t-test. n. Western blot of Ran and RanGAP1 in 20 µg total protein from LOX cells or TMVs. Representative blots (N=3 biologically independent experiments) shown. For all panels, P-values ≤0.05 were considered significant. Unprocessed blot images shown in Supplemental Image 7. Statistical Source in Supplementary Table 1.