*4.4. Expression of Cytochrome P450 Reductase*

Expression of CPR from *Rattus norvegicus* was performed as previously described [41,44].

#### *4.5. Assembly of CYP2C8-Nanodiscs*

CYP2C8-ND were assembled as previously described [37,44,45] by mixing CYP2C8, membrane scaffold protein (MSP1E3D1), an 80:20 ratio of POPC:POPS lipids, and cholate, followed by detergent removal using Amberlite® beads, and purification by size exclusion chromatography [31,32].

#### *4.6. Carbon Monoxide Binding Assay*

The heme content of the purified CYP2C8 proteins was analyzed using UV–vis spectroscopy (Agilent Technologies) as previously described [37].

#### *4.7. Paclitaxel Metabolism*

Samples containing 0.1 μM of CYP2C8-ND (\*1/\*2/\*3/R139K/ K399R) were incubated with CPR (0.3 μM), and PAC (70 μM) in 0.3 mL of 0.1 M potassium phosphate buffer (pH 7.4) for 5 min at 37 ◦C. NADPH (200 μM) was added and the mixture was incubated for 5, 10, and 20 min at 37 ◦C, then quenched with equivolume ethyl acetate. Samples were vortexed and thrice-extracted with ethyl acetate, dried under a stream of N2 gas, and then resuspended in 180 proof ethanol for LC–MS/MS quantification.

#### *4.8. Tandem LC–MS*/*MS for the Quantification of 6*α*-Hydroxypaclitaxel*

Samples were analyzed with the 5500 QTRAP LC/MS/MS system (Sciex, Framingham, MA, USA) in Metabolomics Lab of Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Software Analyst 1.6.2 was used for data acquisition and analysis. The 1200 series HPLC system (Agilent Technologies) includes a degasser, an autosampler, and a binary pump. The LC separation was performed on an Agilent Eclipse XDB-C18 (4.6 × 150 mm, 5 μm) with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetontrile). The flow rate was 0.4 mL/min. The linear gradient was as follows: 0–2 min, 95%A; 8–15 min, 5%A; 15.5–22 min, 95%A. The autosampler was set at 15 ◦C. The injection volume was 5 μL. Mass spectra were acquired under positive electrospray ionization (ESI) with the ion spray voltage at +5000 V. The source temperature was 450 ◦C. The curtain gas, ion source gas 1, and ion source gas 2 were 32, 50, and 65, respectively. Multiple reaction monitoring (MRM) was used for quantitation: Paclitaxel *m*/*z* 854.4 → *m*/*z* 569.2; 6α-hydroxypaclitaxel *m*/*z* 870.4 → *m*/*z* 286.2. Internal standard carbamazepine was monitored at *m*/*z* 237.1 → *m*/*z* 194.0.
