Fig. 4. Identification of actively synthesized proteins in resting and stimulated macrophages. (A) Chemical proteomic approach to identify proteins synthesized upon IF-g stimulation. Newly synthesized proteins are tagged with homopropargylglycine (HPG) and biotinylated via CuAAC click reaction with biotin-azide. Biotinylated proteins are enriched, digested, and identified by MS. (B) HPG incorporation validated via click chemistry with rhodamine-azide and proteins resolved by SDS-PAGE. (C) Proteins which are “Made −Stim” and “Made in +Stim” macrophages across three biological replicates as identified by MS analysis. *− denotes representative bands differentially detected between −Stim and +Stim conditions.