PMC

DAI-induced NF-κB activation is RHIM dependent. (A) HEK239T were transfected with an NF-κB reporter plasmid together with an empty plasmid or increasing doses of the indicated DAI constructs, and were analysed for NF-κB-dependent luciferase activity 24 h later. Data are the mean values±s.d. of three transfection points from one experiment; results are representative of three independent experiments. (B) HEK293T cells were transfected with the indicated Flag–DAI constructs or an empty vector. IPs and XTs were analysed for the presence of endogenous RIP1 by IB. (C) HEK293-TRex cells inducibly expressing the indicated Flag–DAI constructs or an empty vector were treated with doxycycline (Dox; 200 ng/ml) for the indicated times; XTs were analysed by IB. DAI, DNA-dependent activator of IRFs; HEK, human embryonic kidney; IB, immunoblot; IP, immunoprecipitates; NF-κB, nuclear factor-kappa B; RHIM, RIP homotypic interaction motifs; RIP, receptor-interacting protein; VSV, vesicular stomatitis virus; WT, wild type; XT, cell extracts.

The RHIM-containing protein M45 of the MCMV inhibits DAI signalling. (A) HEK293T cells were transfected with VSV–M45₁₋₂₇₇ or an empty vector and the indicated Flag constructs. IP and XT were analysed by IB. (B) HEK239T were transfected with an NF-κB reporter plasmid together with an empty plasmid (e.v) or DAI in combination with increasing doses of the indicated M45 constructs (shown in online) and were analysed for NF-κB-dependent luciferase activity 24 h later. Data are the mean values±s.d. of three transfection points from one experiment; results are representative of two independent experiments. (C,D) HEK293T cells were transfected with the indicated VSV or Flag constructs. IP and XT were analysed by IB. In (D), white and black arrowheads indicate modified and unmodified RIP3, respectively. The asterisks indicate constructs with alanine substitutions of the four amino acids (highlighted in online). DAI, DNA-dependent activator of IRFs; FL, full length; HEK, human embryonic kidney; IB, immunoblot; IP, immunoprecipitates; MCMV, murine cytomegalovirus; NF-κB, nuclear factor-kappa B; RHIM, RIP homotypic interaction motifs; RIP, receptor-interacting protein; XT, cell extracts; VSV, vesicular stomatitis virus.

DAI/ZBP1 contains two RHIM domains that mediate the recruitment of RIP1 and RIP3. (A) Sequence alignment of the RHIM domains of human and murine RIP1, RIP3, TRIF and DAI. The four amino-acid motif crucial for RHIM function is boxed. The black and grey shading indicate more than 60% amino-acid-sequence identity and similarity, respectively. (B,C,E,F,H,I) HEK293T cells were transfected with the indicated VSV or Flag constructs. Immunoprecipitates and cell extracts were analysed by immunoblot. In (F), white and black arrowheads indicate modified and unmodified RIP3, respectively (see text and Fig 3C–E) (D) Domain architecture of RIP1 and RIP3, and schematic views of deletion constructs used in (C), (E) and (F). RHIM^* denotes the construct with alanine substitutions of the four amino acids highlighted in (A). (G) Domain architecture of human DAI and schematic views of the constructs used in this study. RHIM^* denotes the construct with alanine substitutions of the four amino acids highlighted in (A) in the indicated RHIM domains. DAI, DNA-dependent activator of IRFs; DD, death domain; HEK, human embryonic kidney; Hs, human; IB, immunoblot; ID, intermediate domain; IP, immunoprecipitates; KD, kinase domain; Mm, murine; RHIM, RIP homotypic interaction motif; RIP, receptor-interacting protein; VSV, vesicular stomatitis virus; WT, wild type; XT, cell extracts; ZBP1, Z-DNA binding protein 1.

DAI-induced NF-κB activation requires RIP1 and RIP3. (A) HEK293T cells were transfected with 20 nM of the indicated siRNAs and, 36 h later, re-transfected with an NF-κB reporter plasmid together with an empty plasmid or two doses of DAI. NF-κB-dependent luciferase activity was measured 24 h after the second transfection. Data are the mean values±s.d. of three transfection points from one experiment; results are representative of three independent experiments. (B,C) HEK293T cells were transfected with VSV–DAI or an empty vector and the indicated Flag constructs. IP and XTs were analysed by IB. In (B), increasing doses of plasmid are shown. (D) HEK293T cells were transfected with Flag–RIP3 or an empty vector and the indicated VSV constructs. At 24 h after transfection, cell lysates were incubated with or without CIP (10 U per 30 μl, 10 min, 37°C) and analysed by IB. (E) HEK293T cells were transfected with the indicated constructs and analysed by IB. In (C–E), white and black arrowheads indicate modified and unmodified RIP3, respectively. CIP, calf intestinal alkaline phosphatase; DAI, DNA-dependent activator of IRFs; HEK, human embryonic kidney; IB, immunoblot; IP, immunoprecipitates; NF-κB, nuclear factor-kappa B; RHIM, RIP homotypic interaction motifs; RIP, receptor-interacting protein; siRNA, small interfering RNA; XT, cell extracts; VSV, vesicular stomatitis virus; WT, wild type.