**Appendix A**

**Table A1.** Functionality of mutants for the transport of Calcein-AM normalized to the wild-type transporter and pairwise statistical comparison of the raw data.


The raw data has been used for statistical comparisons. Ratio and paired Student's *t*-test was used for all comparisons to wild-type ABCB1 and the Walker B mutants E556/1201Q activities since these positive and negative controls were included in every experiment. Where this was not possible (because of the large the number of mutants tested and when their construction was completed), unpaired Student's *t*-test was applied to the data normalized to one hundred percent wild-type activity (highlighted in yellow). *p* values: \* <0.05, \*\* <0.01, \*\*\* <0.001, \*\*\*\* <0.0001, ns = not significant.

**Table A2.** Functionality of mutants for the transport of BODIPY-verapamil normalized to the wild-type transporter activity and pairwise statistical comparison of the raw data.


The raw data has been used for statistical comparisons. Ratio or paired Student's *t*-test was used for all comparisons to wild-type ABCB1 and the Walker B mutants E556/1201Q as above. Where this was not possible, unpaired Student's *t*-test was applied to the raw data (highlighted in yellow). *p* values: \* <0.05, \*\* <0.01, \*\*\* <0.001, \*\*\*\* <0.0001, ns = not significant.


**Table A3.** Functionality of mutants for the transport of OREGON-GREEN taxol bisacetate normalized to the wild-type transporter activity and pairwise statistical comparison of the raw data.

Unpaired Student's *t*-test on the raw data was used for all comparisons except for Q990A vs. wild-type where a paired Student's *t*-test was applied to the raw data (highlighted in yellow). *p* values: \* <0.05, \*\* <0.01, \*\*\* <0.001, ns = not significant.
