Full-length DinF-BH and NorM-NG were expressed with cleavable hexa- and deca-histidine tags at their C-termini, respectively7 (link),9 (link). Mutations were introduced into the dinF-BH and norM-NG genes by using the QuikChange method (Agilent Technologies) and were confirmed by DNA sequencing7 (link),9 (link). Escherichia coli sBL21 (DE3) ΔacrABΔmacABΔyojHI cells34 (link) were transformed with pET-15b vector containing the inserted genes encoding the DinF-BH and NorM-NG mutants.
The membrane expression levels of DinF-BH and NorM-NG were not affected by the mutations described in this study, as judged by western blot using an antibody against the His-tag (Supplementary Fig. 5 ). For the western blot, the antibody (Qiagen #34460) was diluted 2,500-fold before being mixed with the transfer membranes, and each sample examined on the gel was derived from cell membranes isolated from 80 µg E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells expressing the MATE variants.
The membrane expression levels of DinF-BH and NorM-NG were not affected by the mutations described in this study, as judged by western blot using an antibody against the His-tag (
