(a) Reporter gene assay for HEK-293T cells transfected with 50 ng STEEP or empty vector, IFNB1 promoter luciferase reporter, β-actin Renilla reporter, and MAVS or TRIF as indicated for 24 h (n = 3) (ns
P = 0.86, ns
P = 0.23, lift to right). (b) Immunoprecipitation of Flag-tagged STING and STEEP (n = 3 biologically independent experiments). (c) Confocal microscopy of HeLa cells transfected with HA-STEEP and FLAG-STING for 24 h followed by mock treatment or cGAMP stimulation. Representative data from one experiment are shown (n = 3 biologically independent experiments). (d) Alignment of STEEP/CxORF56 from the indicated species, and highlight of residues mutated to alanine in Mut 1-5. Data in panel a are shown as means of biological triplicates +/- st.dev. Statistical analysis of data shown in panel a was performed using two-tailed Student’s t-test.