Electrophoretic mobility shift assay (EMSA) for the interaction between Ethr and etha promoter

Etha promoter DNA with/without biotin-labeling for the DNA-binding activity assays were synthesized by annealing the complementary single-stranded oligo synthesized by Sangon Co., Ltd. (Sangon, Shanghai, China), and then purified by DNA purification Kit (Tiangen, Beijing, China). Ethr (0.43 μM) was incubated with 0, 2, 5, 10, 25, 50, 100, and 200 μM c-di-GMP and 200 μM c-di-AMP and 200 μM cGMP in a total volume of 13 μL PBS buffer at RT for 1 h. Then biotinylated etha promoter was added and co-incubated at RT for 20 min in a total volume of 20 μL EMSA buffer. The reaction mixtures were then subjected to 8% native PAGE, and transferred to a nylon membrane. The membrane was exposed to 254 nm UV light with 120,000 μJ from HL-2000 Hybridization Incubator (UVP, California, US) for 2 min. EMSA assay was completed using EMSA kit (Thermo) according the manufacturer’s instruction. Images were acquired using a Typhoon Scanner (GE Healthcare).