*2.6. Measurement of Vmax, Km, and CLint*

For preparation of hepatic and intestinal microsomes, the experimental processes were similar to a previously reported method [23,24]. Protein concentration in the hepatic and intestinal microsomes was measured using the bicinchoninic acid (BCA) assay. The in vitro metabolic system consisted of microsomes (equivalent to 1 mg protein); 5 µL dimethylsulfoxide containing final tofacitinib concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 300, and 400 µM; and an nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-generating system (Corning Inc., Corning, NY, USA). The volume of the system was adjusted to 1 mL by adding 0.1 M phosphate buffer (pH 7.4), and the components were incubated in a water-bath shaker at 37 ◦C with 50 oscillations per min (opm) for 15 min. After this incubation, the reaction was terminated by adding two volumes of acetonitrile. Subsequently, two 50-µL aliquots of each reaction mixture were collected. The kinetic constants, maximum velocity (*V*max) and apparent Michaelis–Menten constant (*K*m; the concentration at which the rate is one-half of *V*max for the metabolism of tofacitinib) were determined using the Lineweaver–Burk plot [24,25]. The intrinsic clearance (CLint) for the metabolism of tofacitinib was determined by dividing *V*max by *K*<sup>m</sup> [24,25].

### *2.7. Immunoblot Analysis*

For immunoblot analysis, microsomal protein samples (20–40 µg protein per lane) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and the loaded gel was transferred onto a nitrocellulose membrane for 1 h. For immunodetection, blots were incubated overnight with a primary antibody diluted in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBS-T) containing 5% bovine serum albumin (1:2000) at 4 ◦C with gentle shaking. Subsequently, blots were incubated with secondary antibody conjugated to horseradish peroxide diluted at 1:10,000 with TBS-T containing 5% skim milk for 1 h at room temperature. Protein expression was measured by enhanced chemiluminescence (Bio-Rad) using an Image Quant LAS 4000 Mini (GE Healthcare Life Sciences, Piscataway, NJ, USA). β-actin was used as the internal standard [26]. The density of bands was quantified using ImageJ 1.45s software (NIH, Bethesda, MA, USA).
