Fig 2: Specific IL-1RAcPb mutants gaining the NF-?B signaling functionEach of Swap4567-568, Swap4567-end mutants, IL-1RAcP-WT, or empty constructs was co-transfected with luciferase reporter genes into 293T-IL-1RAcP-KO cells. Then cells were stimulated with titrated concentrations of 5nM IL-1ß for 7 h prior to cell lysis. Finally, the NF-?B activity was measured. Data are shown as means ± SD (n = 3). Significant differences between negative control (NC) group and the other groups were established by Student’s t test. ****p < 0.0001. The protein levels of various IL-1RAcP were measured by Western blot analysis with the whole-cell lysate. The experiments were performed independently at least three times.

Fig 3: Measurement of effects of IL-1RAcP single-site mutations in Swap4/Swap5/Swap6/Swap7 regions of its TIR domain on NF-?B activation signal(A and B) (A) A series of IL-1RAcP single-site mutants with swapped amino acid residues in Swap4/Swap5/Swap6/Swap7 regions of the TIR domain stemming from the corresponding sites of IL-1RAcPb were generated. Each of these mutants, IL-1RAcP-WT or empty constructs was co-transfected with luciferase reporter genes into 293T-IL-1RAcP-KO cells. Then, cells were incubated with titrated concentrations of 5 nM IL-1ß for 7 h prior to cell lysis. Finally, the NF-?B activity was measured. All values represent means ± SD (n = 3). Significant differences between IL-1RAcP-WT group and the other groups were established by Student’s t test. *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001. The protein levels of various IL-1RAcP were measured by Western blot analysis with the whole-cell lysate. The experiments were performed independently at least three times. The mutation sites in IL-1RAcP TIR domain that significantly impaired the NF-?B activation are listed in (B).

Fig 4: Genomic organization, protein architecture of IL-1RAcP isoforms, and measurement of effects of their C-terminal tail mutants on NF-?B activation signal(A) A schematic representation of intron-exon map of human IL-1RAcP locus, alternative splicing, and subsequent translation that leads to the production of protein isoforms. Exons (3–12) encode the mature IL-1RAcP proteins, and differentiated utilization of exon 12 results in two different isoforms, which are distinguished in their TIR domains and C-terminal tails.(B) Detection of the effects of IL-1RAcP and IL-1RAcPb C-terminal tails on NF-?B signaling. Each of IL-1RAcP-?C, IL-1RAcP + bC, IL-1RAcPb-?C mutants, IL-1RAcP-WT, IL-1RAcPb-WT, or empty constructs was co-transfected with luciferase reporter genes into 293T-IL-1RAcP-KO cells (with endogenous expression of IL-1RI). Then, cells were incubated with titrated concentrations of 5 nM IL-1ß for 7 h prior to cell lysis. Finally, the NF-?B activity was measured by dual-luciferase reporter assay. All values represent means ± SD (n = 3). Significant differences between IL-1RAcP-WT group and the other groups were established by Student’s t test. **p < 0.01, ****p < 0.0001. The protein levels of various IL-1RAcP were measured by Western blot analysis with the whole-cell lysate. The experiments were performed independently at least three times.

Fig 5: Sequence alignment of IL-1RAcP and IL-1RAcPb, division of swap regions of their TIR domains, and measurement of the effects of a series of IL-1RAcP swapping mutants on NF-?B activation signal(A) Alignment of C termini of IL-1RAcP and IL-1RAcPb isoforms. Stars (*) indicate conserved residues between IL-1RAcP and IL-1RAcPb. Specific structural or signaling-associated motifs are indicated.(B) Division of Swap regions in IL-1RAcPb-TIR based on sequence alignment and structural analysis. aB to aE of IL-1RAcPb-TIR are marked as Swap1 to Swap9 successively, and labeled by distinct colors. The Swap regions of IL-1RAcP-TIR and IL-1RAcPb-TIR are also shown in (A).(C) Detecting the effects of a series of IL-1RAcP swapping mutants on NF-?B signaling. A series of IL-1RAcP mutants with swapped sequence (from Swap1 to Swap9) stemming from the corresponding regions of IL-1RAcPb were constructed. Each of these mutants, IL-1RAcP-WT or empty constructs was co-transfected with luciferase reporter genes into 293T-IL-1RAcP-KO cells. Subsequently, cells were stimulated by titrated concentrations of 5nM IL-1ß for 7 h prior to cell lysis. The NF-?B activity was then measured. Data are shown as mean ± SD (n = 3). Significant differences between IL-1RAcP-WT group and the other groups were established by Student’s t test. *p < 0.1, **p < 0.01, ***p < 0.001, ****p < 0.0001. The protein levels of various IL-1RAcP were measured by Western blot analysis with the whole-cell lysate. The experiments were performed independently at least three times.