*2.1. Materials*

COS-7 cells and HEK293 cells were purchased from ATCC (Manassas, VA, USA). [3H]- labeled estrone sulfate (ES) and [3H]-labeled p-aminohippuric acid (PAH) were ordered from PerkinElmer (Waltham, MA, USA). Mouse anti-Myc antibody (9E10) was purchased from Roche (Indianapolis, IN, USA). Mouse anti-E-Cadherin antibody was from Abcam (Cambridge, MA, USA). Streptavidin agarose resin, protein G agarose, and Sulfo-NHS-SSbiotin were ordered from Thermo Scientific (Rockford, IL, USA). The 20S proteasome assay kit was ordered from Cayman Chemical Company (Ann Arbor, MI, USA). Mouse anti-βactin antibody, normal mouse IgG, and mouse anti-ubiquitin antibody were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Ixazomib, oprozomib, and delanzomib were purchased from Selleck Chemicals (Houston, TX, USA). Probenecid, lactacystin, epoxomicin and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

### *2.2. Cell Culture*

Parental COS-7 and parental HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 ◦C in 5% CO2. Human OAT3expressing (hOAT3) COS-7 cells and hOAT3-expressing HEK293 cells were established in our group [37,38]. The hOAT3 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 0.2 mg/mL G418 sulfate (Gibco, Grand Island, NY, USA).

## *2.3. Transport Measurement*

The transport activity was assayed using the method published by our lab [30]. Cells per well were incubated in uptake solution of [3H]ES (250 nM) or [3H]PAH (20 μM) in phosphate-buffered saline (PBS)/Ca2+/Mg2+ (PBS/CM) for 3 min. After discarding the uptake solution, the cells were washed twice with cold PBS, then lysed in NaOH solution (0.2 N) and neutralized by adding HCl solution (0.2 N). The amount of ES or PAH uptake was assayed using a Beckman LS 6500 liquid scintillation counter.

#### *2.4. 20S Proteasome Activity Assay*

After incubation with ixazomib, oprozomib, delanzomib, or lactacystin for 6 h, hOAT3 cells were washed once with assay buffer (200 μL) and solubilized in lysis buffer (100 μL). Then, the supernatant (90 μL) was removed to a black 96-well plate, and incubated with SUC-LLVY-AMC solution (10 μL) for 1 h at 37 ◦C. Fluorescence intensity per well (excitation = 360 nm, emission = 480 nm) was assayed using a Molecular Devices Spectramax M3 microplate reader.
