Identification of palmitoylated proteins in DC2.4 cells and MEFs. A) Schematic depicting alk-16 chemical reporter metabolic labeling of live cells and subsequent reaction of cell lysates with detection tags for fluorescence visualization of palmitoylated proteins or affinity purification and proteomic identification of palmitoylated proteins. B, C) Mock treated or IFNα treated DC2.4 cells and MEFS were labeled with alk-16 or DMSO as a control. Lysates were subjected to click chemistry with azido-rhodamine (az-rho) (B) or azido-biotin (C). B) Proteins were separated by SDS-PAGE for fluorescence gel scanning to visualize palmitoylome profiles. Anti-IFITM3 Western blotting served as a control for the activity of IFNα and anti-GAPDH blotting served as a loading control. The red arrow points to the most prominent change in the banding pattern when comparing mock and IFNα treated samples. C) Alk-16/azido-biotin labeled proteins enriched using streptavidin agarose were separated by SDS-PAGE, and were stained with coomassie blue for visualization. Each lane was cut into slices for trypsin digestion and extraction of peptides for MS/MS identification. D) Venn diagram representing proteins identified in C. After subtracting proteins identified in control lanes, 237 DC2.4 proteins and 438 MEF proteins were considered putative palmitoylated proteins. The majority of identified proteins were known to be palmitoylated, including most proteins identified in both cell types and also many of those specifically identified in either DC2.4 cells or MEFs (examples of each are shown in black text). The known IFN-induced and palmitoylated proteins IFITM3 and IRGM1 (blue text) were identified as upregulated in both IFNα-treated DC2.4 cells and MEFs. CD86 and TLR2 are example immunomodulatory proteins specifically identified in DC2.4 cells, while NEDD4 is an example protein specifically identified in MEFs (red text). DMSO, dimethyl sulfoxide; IFITM3, interferon (IFN)-induced transmembrane protein 3; IRFM1, immunity related GTPase M1; MEFs, murine embryonic fibroblasts; MS/MS, tandem mass spectrometry; TLR2, Toll-like receptor 2.