Immunoprecipitation, pull-down, and immunoblotting

After sorting, 2 × 10⁷ pDCs were activated with polyU, and washed with PBS. Cells were lysed in lysis buffer consisting 0.3% CHAPS or 0.3% CHAPS, and 1% IGEPAL CA-630 (Nonidet P-40; Sigma-Aldrich), 40 mM Hepes (pH 7.4), 120 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 1x Halt Phosphatase Inhibitor Cocktail, EDTA free complete protease inhibitor cocktail tablet. For pull-down experiment, 2% glycerol was added. After incubation for 30 min on ice, lysates were centrifuged at 14,500 rpm for 20 min and debris was removed. The N-hydroxysuccinimide-activated Sepharose 4FF beads coupled with anti-GFP antibody, or anti-TLR7 antibody were used for immunoprecipitation. Magnetic Protein G beads, Dynabeads (Invitrogen), or FG beads (Tamagawa Seiki Co.) were also used for immunoprecipitation. For pull-down assay, RalGDS RBD beads (CELL BIOLABS, Inc) was used. Cell lysates were rotated with these beads for 2 h at 4 °C. Beads were washed with the lysis buffer once and washing buffer (0.3% CHAPS or 0.3% CHAPS and 0.5% Nonidet P-40, 40 mM HEPES (pH 7.4), 120 mM NaCl) three times. The bound proteins were subjected to SDS–PAGE. Separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes and detected by immunoblotting with Can Get Signal (TOYOBO).

The AP-MS analyses are described in detail in the extended experimental procedures. Full-length uncropped blots are presented in Supplementary Figs. ^{13– 15}.