TLR2 S-palmitoylation is necessary for NF-kB-dependent gene induction and cytokine production in response to TLR2 microbial ligands. A) TLR2 S-palmitoylation is inhibited by 2-BP in BMDCs. Cells were treated with 100 uM 2-BP or an equivalent volume of DMSO for eight hours prior to labeling for one hour with 50 uM alk-16. TLR2 was immunoprecipitated from cell lysates and reacted with az-rho for fluorescence visualization of palmitoylation. Western blotting with anti-TLR2 antibodies was performed to confirm comparable protein loading. B) Cytokine secretion in reponse to TLR2 ligands is inhibited by 2-BP in BMDCs. Cells were treated with 100 uM 2-BP or an equivalent volume of DMSO for eight hours. TLR2 ligands, Pam3CSK4 (2 ug/mL) or lipomannan (4 ug/mL), or Sendai virus (SeV) (MOI 5), were added to the cellular media for an additional six hours. IL-6 and TNFα levels in cellular supernatants were measured by ELISA. Results in A,B are representative of three experiments. Error bars in B are the standard deviation of triplicate samples. C) Palmitoylation-deficient TLR2 is less able than WT to induce NF-κB-dependent gene expression in response to microbial ligands. HEK293T cells were co-transfected with the indicated plasmids along with a reporter construct expressing NF-κB-driven firefly luciferase and a plasmid constitutively expressing renilla luciferase for normalization. Cells were mock treated or treated for eight hours with Pam3CSK4 (2 ug/mL), lipomannan (4 ug/mL) or zymosan (10 ug/mL), and luciferase activity was measured in cell lysates. Results are presented as fold induction over mock treated samples. D) Western blots confirming similar expression of TLR2-YFP and TLR2-C609A-YFP in experiments performed in C. Results in C, D are representative of at least five experiments. Error bars in C represent standard deviation of triplicate samples. *P <0.001 by Student’s t-test. az-rho, azido-rhodamne; BMDCs, bone marrow dendritic cells; DMSO dimethyl sulfoxide; TLR, Toll-like receptor; 2-BP, 2-bromopalmitate.