*2.6. Immunoprecipitation*

The hOAT3 ubiquitination was investigated using the method published by our group [39]. The hOAT3 cells were lysed in lysis buffer consisted of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mM EDTA, 1 mM NaF, 20 mM N-ethylmaleimide, and 1% of proteinase inhibitor cocktail. Cell lysates were precleared with protein G agarose to decrease nonspecific binding at 4 ◦C for 2 h. Anti-Myc antibody was mixed with protein G agarose (30 μL) and incubated at 4 ◦C for 2 h. The precleared protein was then added to antibody-bound protein G agarose suspension and mixed with end-over-end rotation at 4 ◦C overnight. Proteins coupled to protein G agarose were released with urea buffer containing β-mecaptoethanol and detected by immunoblotting using the anti-ubiquitin antibody.

#### *2.7. Degradation Assay of OAT3*

The hOAT3 degradation was investigated using the method utilized in our group [38]. The hOAT3 cells were first labeled with sulfo-NHS-SS-biotin, then the biotinylated cells were treated with vehicle, ixazomib, oprozomib, or delanzomib at 37 ◦C for 0, 3, and 6 h. Then the cells were collected, and the undegraded cell surface hOAT3 was isolated and detected following the procedures in Section 2.5.

#### *2.8. Electrophoresis and Immunoblotting*

The electrophoresis and immunoblotting experiments were carried out using the method published by our group [30]. Protein samples were loaded on 7.5% precast polyacrylamide gels and transferred onto polyvinylidene difluoride membranes. The immunoblot membranes were blocked with 5% nonfat dry milk in PBS-0.05% tween 20 for 1 h, and incubated with primary antibodies at 4 ◦C overnight, followed by incubation of horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized using a SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific, Rockford, IL, USA), and corresponding densities were analyzed using the FluorChem 8000 imaging system (Alpha Innotech Corp., San Leandro, CA, USA).
