Protein stability analysis by cycloheximide chase assay

Stability of protein was performed by using cycloheximide to treat the cells at several time points to stop protein synthesis as described⁷⁶. Briefly, stable cell lines expressing wild type and mutants-ABCG2 transporter were seeded in 24 well with 1 ml of DMEM (10% FBS, 1.5 mg/ml G418) and cultured for overnight. After remove medium, cells were treated with cycloheximide (Sigma-Aldrich, St. Louis, MO, USA) at final concentration 100 µg/ml (containing 0.1% DMSO) for 0, 1, 3, 6, 12 and 24 h. by using 0.1% DMSO treatment as a control. All samples were collected at the same time and harvested by centrifugation at 15,000 g for 1 min at 4 °C. Cell pellet was lysed in protein lysis buffer containing freshly added protease inhibitor cocktail before perform western blot analysis with mouse anti-ABCG2 (BXP-21) or rabbit anti-β-actin (D6A8) as procedure described above. Quantification used the Image Studio software ver 2.1 (LI-COR^® Biosciences, Homburg, Germany). Regions of measurements for both ABCG2 and β-actin were selected.