Extended Data Fig. 5: DAPK3 does not directly phosphorylate STING or TBK1.
From: The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING–IFN-β pathway
a-d, (a) qRT-PCR of Sting1 and Dapk3, (b) immunoblot, (c) IRF3 nuclear translocation, and (d) p65 nuclear translocation in L929-mRuby-hIRF3 transduced with indicated shRNA stimulated with poly (dA:dT) (0.5 μg/ml) or VACV70 (2 μg/ml) for 3 h. e, Immunoblot of L929-mRuby-hIRF3 transfected with indicated siRNA. f, Immunoblot of L929-mRuby-hIRF3 transduced with indicated shRNA stimulated with VACV70 (2 μg/ml) for 2 h and 4 h. g, Immunoblot of HUVEC stably expressing V5-tagged DAPK3(D161A), DAPK3(T180A), or luciferase. Cells were infected at MOI = 5, 2, or 1. h, Immunoblot of THP1-Blue ISG stably expressing V5-tagged DAPK3(WT) or DAPK3(D161A). i, qRT-PCR of Ifnb1 in L929-mRuby-hIRF3 pre-treated with DAPK inhibitors for 3 h prior to 2′,3′-cGAMP stimulation (10 μg/ml) for 4 h. j, qRT-PCR of IFNB1 in THP1-Blue ISG pre-treated with DAPK inhibitors (50 μM) for 6 h prior to 2′,3′-cGAMP or c-di-GMP stimulation (10 μg/ml for both) for 4 h. k, l, In vitro kinase assay of (k) GST-tagged human STING C-terminus (aa 149-379) and (l) GST-tagged human TBK1(K38M). Peptides were incubated with GST-tagged DAPK3 or TBK1 in the presence of [γ-32P] ATP. Data in (b, e-h, k, l) are representative or (a, c, d, i, j) mean of three independent experiments. Values represent mean ± s.d. *P < 0.05, **P < 0.01, and ***P < 0.001. Statistical comparisons were conducted using two-tailed t-test (a, c, d, i, j).
