Fig. 3: The in vivo photo-crosslinked products corresponding to MacA-MacB complexes of MacA/MacB Bpa variants were significantly reduced after erythromycin treatment.

a The positions of introduced Bpa in MacA and MacB are shown by black arrows in the MacAB-TolC cryo-EM model (PDB: 5NIK). b, c Immunoblotting results of the in vivo photo-crosslinked products of the MacA/MacB Bpa variants. The cells expressing indicated proteins were irradiated for 10 min and analyzed by anti-His tag antibodies. The bands representing MacA-MacB complexes were indicated by black asterisks. All immunoblotting results were determined at least three times independently with similar results. d, e Erythromycin treatment reduced the formation of MacA-MacB complexes in vivo. The cells expressing MacA, MacB^L495Bpa, and TolC, and the cells expressing MacA^V269Bpa, MacB, and TolC were treated at OD₆₀₀ of 1 with 40 µg/ml erythromycin, respectively. After that, cells were irradiated by UV for 20 min and analyzed by anti-his tag antibodies. The expression of EF-Tu was detected by anti-EF-Tu antibodies as the loading control. T-test was used for statistical analyzes. Data are shown as means ± SEM; n = 3. *P < 0.05, **P < 0.01. Ery, erythromycin.