De-myristoylation of ARF1 Protein

De-myristoylation of ARF1 protein by DmX_Vv was examined as described elsewhere with slight modification (Burnaevskiy et al., 2013 (link)). Briefly, HEK 293T cells were transfected with ARF1-strep construct and then myristic acid alkyne (Cayman Chemicals) was metabolically incorporated for 20 hrs. ARF1-strep protein was pulled down using Strep-Tactin Superflow resin (Quiagen) and then treated with wild-type or C3751A mutant GST-DmX_Vv, which were pre-incubated with Δ17ARF1 protein, at 37°C for 30 min. Then, proteins were labeled with azide-fluor 488 (Sigma Aldrich) using Click chemistry reagents as described previously (Charron et al., 2009 (link)). Resulting protein samples were separated on 16% Tricine gel and fluorescence from labeled proteins was captured with a VersaDoc gel document system (Bio-Rad). The same gel was then stained with Coomassie Blue to show equal amount of protein loading.