Extended Data Fig. 10: Systematic screen to make SMART-IL-1β.

a, The X-ray crystal structure (PDB: 4DEP) of interleukin 1β (IL-1β) bound to interleukin 1 receptor, type 1 (IL-1R1). b, IL-1β was split at various sites and the cognate pairs used to generate FLAG-IL-1βN^1-x-eNrdJ-1N^cage-FKBP and FRB-eNrdJ-1C^cage-IL-1βC^y-153–Myc, with x and y respectively denoting the last and first residue of the two fragments (i.e., the split site). Shown is the X-ray crystal structure (PDB: 9ILB) and primary structure of IL-1β with tested split sites indicated. c, Schematic of the in vitro screen used to identify the optimal split site that would render a conditional version of IL-1β: (i) The screen relied on chemically induced FKBP-rapamycin-FRB heterodimerization to trigger conditional protein splicing (CPS) with analysis by Western blotting to identify spliced IL-1β; (ii) spliced IL-1β variants were subsequently tested on cells to determine biological activity using immunofluorescence imaging to determine NF-κB localization. d, Western blot of reactions performed with FLAG-IL-1βN^1-x-eNrdJ-1N^cage-FKBP (0.5 μM), FRB-eNrdJ-1C^cage-IL-1βC^y-153–Myc (0.5 μM) and rapamycin (10 μM) for 2 hr at 37 °C. The expected mass of spliced IL-1β is 20.5 kDa. e-f, Additional experiments for the two pairs FLAG-IL-1βN^1-44-eNrdJ-1N^cage-FKBP/FRB-eNrdJ-1C^cage-IL-1βC^45-153-Myc and FLAG-IL-1βN^1-69-eNrdJ-1N^cage-FKBP/FRB-eNrdJ-1C^cage-IL-1βC^70-153-Myc to validate splicing of IL-1β being rapamycin- and CPS-dependent (the latter experiment used the splicing-deficient eNrdJ-1N(C1A)^cage mutant). g, Cultured HeLa cells were incubated with recombinantly expressed IL-1β for 20 min at 37 °C to induce IL-1R1 signaling and NF-κB nuclear localization. Cells were then fixed (5% formaldehyde), permeabilized (0.5% Triton X100), and analyzed by confocal imaging using a primary rabbit anti-NF-κB (p65) antibody and a secondary goat anti-rabbit antibody Alexa Fluor 488 conjugate. Scale bar equals 100 μm. Subsequent experiments followed a similar analytical procedure. h, Cultured HeLa cells were treated with reaction mixtures of the pair FLAG-IL-1βN^1-44-eNrdJ-1N^cage-FKBP/FRB-eNrdJ-1C^cage-IL-1βC^45-153-Myc and analyzed as described above to determine IL-1R1 signaling activation. Scale bar equals 50 μm. i, HeLa cells were left untreated or incubated with recombinant IL-1β (0.5 nM). In experiments with SMART-IL-1β (i.e. IL-1βN/IL-1βC), HeLa cells were treated with a medium produced by incubating OE19 cells (HER2^high, EGFR^low, EpCAM^high) with IL-1βN-αHER2 (20 nM) and αEpCAM-IL-1βC (20 nM) for 2 hr at 37 °C. In all cases, HeLa cells were incubated for 30 min at 37 °C, before being collected and analyzed by Western blotting to determine the phosphorylation of NF-κB (pNF-κB) and IκB (pIκB). j, The localization of NF-κB was examined for OE19 cells untreated or incubated with recombinant IL-1β. Scale bar equals 40 μm. Data shown are representative of two independent experiments.