Protein Palmitoylation Detection Protocol

Studies of protein palmitoylation were performed according to detailed published protocols[95 (link)–97 (link)]. In brief, transfected cells were treated for 1 h with the alk-16 (20 uM) chemical reporter of protein palmitoylation. Proteins of interest were immunoprecipitated from cell lysates and reacted via the copper(I)-catalized azide alkyne cycloaddition reaction (“click chemistry”) with azido-rhodamine (kindly provided by Dr. Howard Hang of the Rockefeller University) for visualization of protein acylation via fluorescence gel scanning on a Typhoon 9400 (Amersham) fluorescence imager. Western blotting of the samples provided controls for loading and sample inputs. For inhibition of protein palmitoylation, transfected cells were treated for 24 h with 100 uM 2-bromopalmitate (Sigma).