*4.2. Cloning of ABCG5*/*G8 Missense Mutants*

The expression vectors (pLIC and pSGP18), carrying human ABCG5 (NCBI accession number NM\_022436) and human ABCG8 (NCBI accession number NM\_022437), were derived from pPICZB (Invitrogen) as described [33,45], pLIC-ABCG5, and pSGP18-ABCG8, respectively. A tandem array of six histidines separated by glycine (His6GlyHis6) was added to the C terminus of ABCG5, and a tag encoding a rhinovirus 3C protease site followed by a calmodulin binding peptide (CBP) was added to the C terminus of ABCG8. To generate the missense mutants in this study, we performed site-directed mutagenesis by using WT ABCG5 or ABCG8 as the templates and the following codon-optimized oligonucleotide primers (Eurofins Genomics Canada). G5-A540F: CCATTTTTGGGGTGCTTGTTGGATCTGGATTCCTCAG (forward) and GCACCCC AAAAATGGACAGCAGAGCCACTACAC (reverse); G5-E146Q: GCGCCAAACGCTGCACTACACC GCGCTGC (forward) and CAGCGTTTGGCGCACGGTGAGGCTGCTCAG (reverse); G8-R543S: GTT GCTCTATTATGGCCCTGGCCGCCGC (forward) and GCCATAATAGAGCAACAGAAGACCACCAG CCAC (reverse); G8-G216D: ACGAGCGCAGGAGAGTCAGCATTGGGGTGCAG (forward) and CTCTCCTGCGCTCGTCCCCCGACAACCCC (reverse). The polymerase chain reaction (PCR) included 1× Phusion High-Fidelity DNA Polymerase (New England Biolabs), 1× Phusion buffer, 200 mM dNTP, 2% (*v*/*v*) DMSO, 100 ng DNA templates, and 0.4 mM forward and reverse primers. Each mutant-containing plasmid was amplified by the following PCR settings: initial DNA denaturation (98 ◦C, 2 min), followed by 30 cycles of denaturation (98 ◦C, 15 s)/primer annealing (55 ◦C, 30 s)/DNA extension (72 ◦C, 3 min), and then final extension (72 ◦C, 20 min). Next, 5 µL of the PCR products was run on a 1% agarose gel to confirm the amplification, and 1 µL of *Dnp*I restriction enzymes (20 units) was used to digest the WT templates overnight at 37 ◦C. The modified plasmids were cleaned up using the ethanol acetate precipitation technique. Then, 5 µL of 3 M sodium acetate was added to each 50 µL PCR product. Next, 200 µL of 100% ethanol was added to each tube, vortexed, and left at room temperature for 10 min. At max speed in a table centrifuge for 10 min, the plasmids were pelleted; then, the supernatant was removed and washed with 75% ethanol. Residual ethanol was dried by a Speed-Vac at the maximal speed for 20 min at room temperature. The pellet was resuspended in ddH2O. Mutants plasmids were cloned into XL1-Blue competent *E. coli* cells by the heat-shock approach as described in the supplier's manual (Novagen/Agilent, Santa Clara, CA, USA) and by antibiotic selection using Zeocin (Invitrogen/ThermoFisher, Ottawa, ON, Canada). Using PureYield Plasmid Midiprep kit (Promega, Madison, WI, USA), DNA preparations of selected clones were subjected to sequencing at Eurofins Genomics Canada.
