Confirmation of the S-palmitoylation of immunomodulatory proteins, CD86 and TLR2. A-C) Validation and mapping of CD86 and TLR2 palmitoylation. HEK293T cells were transfected with the indicated CD86-HA (A), FLAG-TLR2 (B), or TLR2-YFP (C) constructs. Transfected cells were labeled with 50 uM alk-16 or DMSO as a control for one hour. Cell lysates were subjected to immunoprecipitation for the respective epitope tag or fluorescent protein. Immunoprecipitates were reacted with azido-rhodamine (az-rho) for visualization of palmitoylation by fluorescent gel scanning. Western blotting was performed to confirm comparable protein loading. D,E) Overexpression screens to identifiy DHHCs capable of modifying CD86-HA and FLAG-TLR2. HEK293T cells were co-transfected with HA-tagged DHHCs 1 to 23 or GST as a control and either CD86-HA (D) or FLAG-TLR2 (E). Immunoprecipitated proteins were treated and visualized as in A-C. Western blotting was performed to confirm comparable protein loading. Bar graphs represent average quantified fluorescence intensities from at least three identical experiments normalized to their respective western blots. Values for CD86 or TLR2 co-transfected with GST control were set to 1. DHHC constructs are labeled according to previously used DHHC nomenclature in order to be consistent with past studies using these constructs. Importantly, DHHCs 2, 3, 6, 7 and 15 are the same in both the previous and modern nomenclature. Note that in D, DHHC14 itself is visible in the fluorescent gel scan of this region. The expression levels of the individual DHHCs obtained in a representative experiment are shown in Additional file 2: Figure S6. DMSO, dimethyl sulfoxide; TLR2 Toll-like receptor 2.