Recombinant mouse ifn β

Osteoclastogenesis assay was performed with RAW 264.7 cells (1.5*10⁴ cells per well in a 24-well plate) that were incubated with 30 ng/ml RANKL (Peprotech, Israel) for 5 days. RANKL-treated cells were also treated with Zol and/or Dex, recombinant mouse IFN-β (0.25 or 2.5 ng/ml, Biolegend), or anti-IFN-β antibodies (2 μg/ml, Abcam, United Kingdom) for the first 2 days of incubation and then washed. RANKL was supplemented after washing.

pMEFs were generated from six different WT and SK1^−/− embryos and frozen stocks generated at passage 3. Mice were used in accordance with animal ethics 181/12 approved by the SA Pathology/CALHN animal ethics committee. All MEF cultures were used in infection studies within 2 weeks of thawing. Individual studies were verified in at least two different pMEF cultures. Cells were treated in culture for 15–20 min with recombinant mouse IFN-β (Biolegend, San Diego, CA, USA) or pretreated for 90 min with 5 μM SK1-I (Enzo Life Sciences, Farmingdale, NY, USA) prior to stimulation with IFN-β. HEK293 cells expressing control or SK1 shRNA were generated following lentivirus transduction and selection as previously described.^{44 (link)} Cells were passaged in tetracycline-free media (Dulbecco's modified Eagles medium (DMEM) with 10% (v/v) fetal calf serum) to minimise basal shRNA expression.

We generated a mouse monoclonal antibody that recognized an epitope within amino acids 322-339 of human STING and within amino acids 321-338 of mouse STING (Fig. S2A-D). A polyclonal antibody against phospho-S365 of mouse STING was generated in rabbits and affinity-purified (Fig. S2E). Polyclonal antibodies against mouse STING, Igα, Igβ, class I MHC heavy chain, and class II MHC α chain were generated in rabbits. An anti-SEL1L polyclonal antibody was also generated in rabbits and affinity-purified. The following antibodies were purchased: XBP1s (Cell Signaling), HRD1 (Abgent), HRD1 (Proteintech), p97 (Fitzgerald), μ (SouthernBiotech), κ (SouthernBiotech), phospho-Syk (Cell Signaling), Syk (Cell Signaling), phospho-Igα (Cell Signaling), TCL1 (Cell Signaling), cleaved caspase 3 (Cell Signaling), cleaved caspase 7 (Cell Signaling), calnexin (Proteintech), and actin (Sigma). LPS and digitonin were purchased from Sigma. Recombinant mouse IFNβ and TNFα were procured from Biolegend. Kifunensine and MG132 were purchased from Cayman. 3’3’-cGAMP was chemically synthesized as described previously.^{28 (link)}

Naive splenic B cells were purified using a negative selection magnetically assisted cell sorting kit (Miltenyi Biotech) according to the manufacturer’s protocol and cultured in IMDM (Gibco, Invitrogen) containing 10% heat-inactivated FBS (Wisent) and supplemented with penicillin-streptomycin (Life Technologies). Purified B cells were then incubated by themselves, with the parasite at a multiplicity of infection (MOI) of 5 or 10, or with 10 ng/mL mouse recombinant IFN-β (BioLegend) at 37°C and 5% CO₂ for the duration specified. For experiments assessing the necessity of direct contact for B cell activation, transwell permeable membrane inserts (Corning) with a pore size of 0.4 μm and a polyethylene terephthalate (PET) membrane were used to separate total B cell populations or B cell subpopulations, and only the cells in the top compartment were exposed to PKH67-labeled parasite.

Naive splenic B cells were purified using a negative selection magnetically assisted cell sorting kit (Miltenyi Biotech) according to the manufacturer’s protocol and cultured in IMDM (Gibco, Invitrogen) containing 10% heat-inactivated FBS (Wisent) and supplemented with penicillin-streptomycin (Life Technologies). Purified B cells were then incubated by themselves, with the parasite at a multiplicity of infection (MOI) of 5 or 10, or with 10 ng/mL mouse recombinant IFN-β (BioLegend) at 37°C and 5% CO₂ for the duration specified. For experiments assessing the necessity of direct contact for B cell activation, transwell permeable membrane inserts (Corning) with a pore size of 0.4 μm and a polyethylene terephthalate (PET) membrane were used to separate total B cell populations or B cell subpopulations, and only the cells in the top compartment were exposed to PKH67-labeled parasite.