Pull-down assay was performed as described with some modifications (55 (link)). All proteins for in vitro synthesis were produced using the cell-free PURExpress in vitro protein synthesis system (New England Biolabs) in the presence of 0.12 mg/ml proteoliposomes at 37°C for 3 h. Proteoliposomes were prepared using soybean L-α-phosphatidylcholine (Sigma) in buffer (20 mM Tricine, 20 mM succinic acid, 80 mM NaCl and 0.6 mM KOH, adjusted to pH 8.0) to a concentration of 32 mg/ml as described (46 (link)). DNA templates were prepared according to the manufacturer’s instructions. To synthesize UgtL-HA, PhoQ-FLAG, PhoZ-FLAG, and EnvZ-FLAG, we used primers 16061/16062 for UgtL-HA, 16063/16064 for PhoQ-FLAG, 16063/16065 for PhoZ-FLAG, 16065/16066 for EnvZ-FLAG. At the end of the reaction, samples were diluted in TBS buffer (Tris-buffered saline) with 20 times the reaction volumes. Diluted reactions were mixed in 500 μL TBS containing 0.12 mg/ml proteoliposomes and incubated at room temperature for 2 h. Then samples were pulled down with magnetic beads conjugated to antibodies recognizing the HA epitope at 4°C for 1 h or with magnetic beads conjugated to antibodies recognizing the FLAG epitope at room temperature for 2 h. Pulled down samples were analyzed by Western blotting using antibodies recognizing the HA or FLAG epitopes.