Flag m2 bead

HEK293T cells were transfected with HA‐ubiquitin, FLAG, or Myc‐tagged constructs by using polyethylenimine (PEI, Polysciences, 23966). After 24 h later, cells were activated by 200 ng/ml LPS for 4 h followed by 15 μM nigericin for 30–60 min. Cell lysates were immunoprecipitated with Flag‐M2 bead (Sigma‐Aldrich, A2220) or anti‐Myc antibody at 4°C overnight with agitation. Beads were washed with lysis buffer four times. They were eluted with 2× SDS sample buffer, and subjected to western blotting.

Immunoprecipitation was performed following established protocols, as described previously (39 (link)). HeLa cells were seeded in a 12-well plate at a density of 1 × 10⁵ cells per well with 1 mL of complete growth medium, then co-transfected with pCMV-Flag-STING (Sino Biological, China, catalog no. HG29810-NF) and pCMV-HA-Ub (Ke Lei Biotechnology, China, catalog no. kl-zl-0513) for 12 hours followed by R. rickettsii infection at an MOI of 1 for 2 days, and 100 nM MG132 were added to cells for 8 hours before cells were lysed in IP buffer (Beyotime, China, catalog no. P0013). After pre-clearing with protein A/G agarose beads for 1 hour at 4°C, whole-cell lysates were used for immunoprecipitation with the indicated antibodies. A 50% slurry of FLAGM2 beads (Sigma, United States, catalog no. M8823) was added to 1 mL of cell lysates and incubated at 4°C for 6 hours. Immunoprecipitates were extensively washed five times with lysis buffer and eluted with SDS loading buffer by boiling for 5 minutes. The following immunoblot steps were consistent with Western blotting analysis.

For Flag-tagged proteins’ immunoprecipitation experiments, Flag-tagged constructs were transfected into 293T cells, and then cells were harvested 24 h after transfection. Flag M2 beads (Sigma, A2220) were incubated with cell extract at 4 °C for 2 h. For other immunoprecipitation experiments RPE-1 cells were lysed at 4 °C for 30 min with ELB buffer: 50 mM HEPES (pH 7.0), 250 mM NaCl, 5 mM EDTA (pH 8.0), 0.1% NP-40, 10% glycerol, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and phosSTOP. Extracts were cleared by subsequent centrifugations. Immunoprecipitation was carried out with an indicated antibody at 4 °C overnight. Proteins were separated by SDS–polyacrylamide gel electrophoresis and analyzed by western blotting with the indicated antibodies. Images have been cropped for presentation (uncropped scans of the blots were shown in Supplementary Fig. 10). The same results were obtained in at least three separate experiments for each interaction assay.

Collected cells were lysed in RIPA buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.25% sodium deoxycholate and 1 mM EDTA) supplemented with protease inhibitors including 10 μg⁻¹ ml⁻¹ aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, and 10 μM leupeptin. Equivalent proteins were loaded, separated by SDS-PAGE, and detected by immunoblotting using Amersham Imager 600 (GE Healthcare Life Sciences). Uncropped scans of all western blots are shown in the Supplementary Figs. 11–15. For immunoprecipitation, cells were collected and lysed in 1 ml NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM sodium pyrophosphate, 2 mM EDTA, and 5% NP-40, supplemented with protease inhibitors, including 10 μg⁻¹ ml⁻¹ aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, and 10 μM leupeptin) for 10 min. Then, the soluble fraction was isolated by centrifugation at 21,130 × g for 20 min at 4 °C. FLAG M2 beads (Sigma-Aldrich) were incubated with 1 ml cell lysate at 4 °C for 3 h. After incubation, complexes were washed five times with NP-40 lysis buffer and the bead-conjugated proteins were denatured in 2× SDS loading buffer for 15 min at 95 °C. Protein samples were then separated by SDS-PAGE and detected by immunoblotting. Uncropped blots can be found in Supplementary Figs. 11–15.

AML12 cells (ATCC) were grown in DMEM/F12 (Wisent) supplemented with 10% FBS (Wisent), insulin (Sigma), Holo-Transferrin (Sigma), Dexamethasone (Sigma). Cells were stably transfected with either the empty vector (EV) p3X-flag or the p3X-WTmAS3MT-flag constructs and selected with 900 μg/mL of G418 (Wisent). Immunoprecipitation using Flag M2 beads (Sigma) was performed according to manufacturer’s instructions. Beads were washed and sent to MS/MS for analysis at Université de Sherbrooke. The analysis was performed as previously described (Dubois et al. 2016 (link)).