HPG biosynthetic protein labeling and quantitation
Dissected larval brains were incubated in PBS containing 50 µM HPG for three hours. The Click-iT HPG Alexa Fluor 488 Protein Synthesis Assay Kit (ThermoFisher) was used to fluorescently label HPG-containing proteins. The HPG labeling reaction was followed by standard antibody staining to detect Elav protein. Pixel intensity measurements of the HPG-Alexa Fluor 488 signal were made using ImageJ and the “measure” tool applied to an identical size area encompassing multiple neurons per brain, with all staining procedures and confocal settings identical across all samples.