Myristoylation assay

Global myristoylation was measured following a established method⁵³. Briefly, H460 cells grown overnight were washed with PBS to remove serum and pre-incubated for 1hour with Optimem (Thermo Fisher Scientific) containing 1 μM NMT inhibitor or DMSO control. This was then replaced by medium containing 1 μM NMT inhibitor or DMSO control and 25 μM of Alkynyl Myristic Acid (Click Chemistry tools) for 6 h in culture. Protein extracts were prepared using NP40-based lysis buffer and a total of 200 μg of protein per experimental condition assayed in the presence of biotin-PEG3-azide using a Click Chemistry Protein Reaction Kit as indicated by the manufacturer (Click Chemistry Tools). Pellet resulting from the reaction was resuspended in electrophoresis loading buffer, boiled and subjected to electrophoresis. Signal was detected with streptavidin-HRP staining (Cell Signaling Technologies) and developed with Supersignal WestPico PLUS (Pierce).