*4.1. Site-Directed Mutagenesis*

The cDNA encoding ABCB1 including a 12 histidine carboxy-terminal tag was described previously [20]. This cDNA was subcloned into pCI-neo to generate pCI-neo-ABCB1-12his (henceforth designated pABCB1). The coding sequence was modified by lightning site-directed mutagenesis (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer's recommendations except for generation of the Q347A mutant where a

lower annealing temperature of 50 ◦C was required. The individual mutants for Q347A, Q725A, and Q990A were made first followed by the sequential addition of the second and third mutations. As a negative control for the transport experiments, the catalytic glutamates within NBD1 and NBD2 (Glu556 and Glu1201, respectively) were mutated to glutamines, thus preventing activation of the water for nucleophilic attack on the bound ATP. This mutant was generated previously [21]. Being unable to hydrolyze ATP, this mutant becomes trapped in the ATP-bound state [22]. Each cDNA was fully sequenced to ensure veracity.

Mutagenic oligonucleotides with the new alanine codons emboldened (Table 1).


**Table 1.** Mutagenic oligonucleotides with the new alanine codons emboldened.
