STING Ubiquitination Immunoprecipitation

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.