2.4.1. Of the Single Mutants Only Q990A Appears to Reduce the Transport of OG-Taxol

− The Q347A and Q725A mutants were not distinguishable from the wild-type transport activity. However, the reduced activity of the Q990A mutant reaches statistical significance only after the raw data are paired (Figure 5). The effect is subtle with the Q990A mutant retaining 66 ± 8% transport activity for OG-taxol when normalized to wild-type ABCB1.

− ≥ < **Figure 5.** Functionality of glutamine to alanine mutants for the transport of OG-taxol. Live HEK293T cells transiently expressing equivalent amounts of wild-type (wt) and mutant ABCB1 were challenged with OG-taxol. Functionality was measured as the ratio of OG-taxol accumulation between the ABCB1-expressing and untransfected cells within the population. This was normalized to 100% for wild-type ABCB1 for the bar graph shown. The mean ± SEM was plotted using GraphPad Prism version 8; sample number was ≥3. Selected statistical analysis (unpaired Student's *t*-test, two-tailed performed on the raw data except for the comparison of the wild-type with Q990A for which the raw data are paired) is shown with *p* value: \* <0.05. The full pairwise comparison of the data is given in Appendix A Table A3.

2.4.2. The Double Mutants Q347/990A and Q725/990A Reduce the Transport Activity Further but the Triple Mutant Restores Wild-Type Levels of OG-Taxol Transport

The Q347/725A double mutant is trending towards reduced transport of OG-taxol but does not reach statistical significance. However, both Q347/990A and Q725/990A have reduced transport activity for OG-taxol, emphasizing the negative effect of the Q990A mutation. Perhaps surprisingly, given that all pairwise mutants seem to have reduced transport of OG-taxol, the triple mutant restores transport activity to wild-type levels.
