Figure 4

DP71L inhibits STING-TBK1 interaction. A Domain organization of STING, highlighting GST-tagged aa 1–145 (TM), aa 1–185 (both TM and DD), aa 1–340 (TM, DD and CBD) and STING-WT. TBK1 binding motif and TBK1 phosphorylation site on STING CTT are conserved among different species. B HEK293T cells were transfected with GST-tagged STING domain constructs and the DP71L-Strep plasmid. At 36 hpt, WCLs were subjected to a GST PD assay, followed by immunoblotting using the indicated antibodies. C HEK293T cells were transfected with STING-Strep, TBK1-GST, and increasing amounts of DP71L-Flag plasmids. At the 24 hpt, cells were stimulated with 4 µg/mL of cGAMP for an additional 12 h. WCLs were then subjected for a Strep PD assay, followed by immunoblotting with the specified antibodies. D 3D4/21 cells were transfected with increasing amounts of DP71L-Flag plasmids. Subsequently, at the 24 hpt cells were stimulated with 4 µg/mL of cGAMP for an additional 12 h. WCLs were then utilized for the IP using STING antibody, followed by immunoblotting using the designated endogenous antibodies. E HEK293T cells, transfected with STING-Strep expression plasmids and increasing amounts of DP71L-Flag plasmids, were stimulated with 4 µg/mL of cGAMP. After 4 h, cell lysates were separated using SDD-AGE (top) or SDS-PAGE (bottom) and probed with the indicated antibodies. F PK-15 cells were transfected with control or different amounts of DP71L-Flag plasmids (100 and 200 ng). After 24 h, cells were stimulated with 4 µg/mL of cGAMP for an additional 12 h. Subsequently, the cells were fixed with 4% paraformaldehyde and prepared for confocal imaging. In immunoblot data, protein sizes are expressed in kDa. All the data are representative of at least three independent experiments, each with similar results.