Bacterial two-hybrid assay (BTH)

We used BTH assay to test protein–protein interaction in vivo. This method is based on the reconstitution of the adenylate cyclase from Bordetella pertussis⁴¹. Proteins of interest were fused to T18 and T25 fragments of adenylate cyclase using pUT18c and pKT25. After co transformation in BTH101 strain, the plates were incubated overnight at 30 °C. Colonies were re-streaked overnight at 30 °C. Then, 1 mL of LB supplemented with ampicillin and kanamycin was inoculated in the morning and incubated at 30 °C with shaking for 8 h. In total, 5 µl of undiluted cultures were then spotted on NA agar plates supplemented with X-Gal 40 µg/mL with different concentrations of IPTG. For β-galactosidase experiments, the same inoculum has been used, and β-galactosidase activity was determined as described by Miller with the use of the TECAN microplate reader to follow OD₆₀₀ and OD₄₂₀ over time.