Fresh whole cell lysates of ACHN cell lines were used to detect the activity of PROTAC on Smad3 degradation via ubiquitin–proteasome pathway in vitro [50]. The cell lysates were quantified with BCA assay (Thermo Fisher Scientific, USA). Smad3 recombinant protein (HY-{"type":"entrez-protein","attrs":{"text":"P71323","term_id":"75344926","term_text":"P71323"}}P71323, MedChemExpress, USA) and PROTAC in different concentrations (0, 1, 5, 10 μg) were added to the cell lysates in a water bath at 37 °C for 30 min. Then 33 μL of 4 loading buffer was added to 100 μL of this reaction mixture. The sample was boiled for 10 min before western blot analysis. To further explore weather the degradation of Smad3 was through ubiquitin proteasome pathway, 5 μM or 10 μM MG132 (proteasome inhibitor, MedChemExpress, USA) were added into the cell lysates too. Then the protein level of Smad3, VHL and ubiquitin were all detected by western blot.
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