Fig 2: Correlation between Arf6-GTP and luciferase in cell lines producing A350V (A) or wild type (B) IQSEC2 luciferase. Arf6-GTP was measured by ELISA (spectrophotometric units). The correlation between the relative amount of IQSEC2 and Arf6-GTP was significant for A350V (r = 0.77, n = 10, p = 0.009) but not for WT IQSEC2 (r = 0.52, n = 6, p = 0.28).

Fig 3: Silvestrol-sensitive mRNAs are enriched with complex 5′ UTRs. (a) Box plot showing distribution of free energy values for 5′ UTRs of Silvestrol-sensitive genes (Decreased, n =284, z-score below -1.5), genes increased in Silvestrol (n =146 genes, z-score >1.5), and genes insensitive to Silvestrol (n =6,303). ***P =2.53 × 10-67, **P =1.097 × 10-27 and *P =4.25 × 10-7. Energy values were predicted using the CONTRAfold algorithm [25]. (b) Box plot showing distribution of 5′ UTR length, L: ***P =1.39 × 10-133, **P =3.1 × 10-44 and *P =4.83 × 10-10. (c) Box plot showing distribution of GC content: ***P =1.02 × 10-47, **P =3.35 × 10-23 and *P =8.08 × 10-6. For genes with decreased TE: n =284, ∆Gmean = -103.9 kcal/mol, Lmean =493.4 nucleotides, GCmean =59.8%. For insensitive genes: n =6,303, ∆Gmean = -54.72 kcal/mol, Lmean =223.6 nucleotides, GCmean =66.9%. For genes with increased TE: n =142, ∆Gmean = -45.93 kcal/mol, Lmean =171.8 nucleotides, GCmean =71.23%. Significance values were determined using the two-tailed Mann-Whitney U test (d) RPKM and TE values from ribosome profiling data for genes used in 5′ UTR analyses of CyclinD1, ARF6, ROCK1 and PFN2. (e) Luciferase reporter constructs, stably transfected into 293 T cells, were treated with increasing concentrations of Silvestrol and luciferase expression was measured after 40 minutes; constructs bearing 5′ UTRs from Silvestrol-sensitive genes (CyclinD1, ROCK1, ARF6) or insensitive genes (PFN2 or CMV alone) were compared. Triplicate values were obtained in each experiment; data presented were obtained from four independent experiments. (f) Luciferase reporter expression in stably transfected 293 T cells treated with increasing concentrations of Silvestrol; constructs bearing ARF6wt 5′ UTR or ARF6mut 5′ UTR were compared; data presented were obtained from two independent experiments with measurements taken in triplicate.

Fig 4: Show the specificity of the immune-magnetic bead method used to isolate exosomes and microparticles. (A) Show gating strategy of streptavidin-labeled magnetic beads used by flow cytometry using Phycoerythrin (PE)-labeled biotin or Fluorescein isothiocyanate (FITC)-labeled biotin. The forward scatter (FSC), and side scatters (SSC) data were used to gate for a single population of beads for analysis, avoiding clumped beads. (B) Show the percentage of magnetic beads conjugation with streptavidin using FITC-Biotin and PE-Biotin. (C) Validation of immune-magnetic affinity isolation methods using extracellular vesicles isolated from Huh-7.5 cell culture. A very high percentage of exosomes were detected by using CD63 antibody-coated magnetic beads. A very high rate of ARF6 positive microvesicles was seen using Annexin A1 coated beads. (D) Show reproducibility of the assay separating exosomes from microvesicles. The results are expressed as the mean percentage ± standard deviation and analyzed by Mann Whitney U-test. **P < 0.01, ***P < 0.001.

Fig 5: NMT1 and NMT2 have lysine transferase activity and can modify ARF6 on K3.a Lysine steric properties resemble those of N-terminal glycine. b The alignment of the N-terminal sequences of human ARFs reveals a unique lysine residue in ARF6. c Monitoring NMT1 in vitro reactions on different ARF6 peptides using LC-MS. Total ion chromatograms searched for the substrate and product ions are shown. d HPLC separation of NMT reaction on ARF6 WT N-terminal peptide reveals mostly glycine myristoylated product. e NMT reaction with ARF6 WT protein and Alk12-CoA generates several modified species. Shown are in-gel fluorescence and FLAG western blot after TAMRA azide conjugation via click chemistry. f Simulated annealing omit 2FO–FC map (at 1.2 σ) surrounding the myristoyl-KVLSKIF product in NMT2. Peptide: cyan, Lys3: pink, myristoyl: orange. g Myristoyl–peptide and CoA products bound to chain A in the NMT1 myristoyl-AcKVLSKIF structure. The 1.0 σ 2FO–FC electron density map around the ligands is shown (blue mesh). h Comparison with PDB structure 509V (black), NMT1 containing myristoyl-glycine peptide product. i Model showing that NMT1 and NMT2 enzymes can modify G2, K3 of ARF6, or both.