Western Blotting of Organoid Proteins

Western blotting was performed as described previously^{57 (link)}. In brief, organoids were collected and lysed in Pierce^TM RIPA Buffer (ThermoFisher Scientific, Cat. #89900), and a cocktail of protease and phosphatase inhibitors (Roche). Organoids were then sonicated, and protein concentration was quantified using Pierce^TM bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific, Cat. #23227) according to the manufacturer’s instructions. Samples were then heated for 10 min at 100 °C before loading. An equal amount of proteins was loaded and separated using Mini-PROTEAN TGX Stain-Free-Gels (Bio-Rad, Cat. #4568044), separated proteins were then transferred onto iBlot 2 PVDF Mini Stacks (Invitrogen, Cat. #IB24002). The membrane was then blocked with 5% Skim Milk in TBS-T (20 mM Tris–HCl, pH 7.6, 136 mM NaCl, and 0.1% Tween-20) for 1 h at RT, followed by primary antibody incubation diluted in 5% BSA for 12 h at 4 °C. The primary antibodies used in this experiment are listed in Supplementary Table 5. The membrane was then washed three times with 1× TBST for 10 min each at RT before incubation with secondary antibody diluted 1:5000 in 5% Skim Milk in 1× TBST for 1 h at RT. The membrane was washed again three times with 1× TBST for 10 min each at RT before visualization with Clarity Western ECL Substrate (Bio-Rad, Cat. #170-5060).