Figure 3
From: A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation

Phosphorylation of STING by TBK1 in vitro requires “ER-to-Golgi” traffic and palmitoylation of STING. (a) TBK1-knockout MEFs were treated with brefeldin A (3 µg/mL) for 30 min followed by the stimulation with DMXAA for 1 h. The post-nuclear supernatants of cells were centrifuged at 100,000 × g. The resulting membrane fraction was resuspended, incubated with recombinant TBK1 in the presence of ATP, and analyzed by western blot. (b) Post-nuclear supernatants of unstimulated TBK1-knockout MEFs were centrifuged at 100,000 × g. The resulting membrane fraction was resuspended and incubated with recombinant TBK1 and ATP in the presence of DMXAA (25 µg/mL) or 2′3′-cGAMP (250 ng/mL) at 37 °C for 30 min. Phosphorylation of STING at Ser365 was analyzed by western blot. (c) TBK1-knockout MEFs were treated with 2-bromopalmitate (50 µM) for 30 min followed by the stimulation with DMXAA for 1 h. The membrane fraction of the cells was resuspended, incubated with recombinant TBK1 in the presence of ATP, and analyzed by western blot.