Pelleted cells were lysed using RIPA buffer (Life Sciences) using HALT protease inhibitor cocktail (Sigma). Genomic DNA was sheared through a 27G needle, followed by centrifugation to clear cellular debris. Cleared protein lysate concentrations were quantified using BCA protein assay kit (Thermo Fisher).
Flash frozen tissue samples of 50–100 mg were isolated over dry ice and lysed using 100–200 μL RIPA buffer with HALT protease inhibitor cocktail. Tissue homogenization was conducted using a microtube homogenizer (Cole-Parmer), followed by centrifugation and quantification of cleared protein lysate as above.
Organoid samples were digested from reduced growth factor Matrigel in 10mM EDTA in PBS for 1 hour at 4°C, After centrifugation and resuspension in 20–40 μL RIPA buffer, protein lysate samples were cleared and quantified as above.
Flash frozen tissue samples of 50–100 mg were isolated over dry ice and lysed using 100–200 μL RIPA buffer with HALT protease inhibitor cocktail. Tissue homogenization was conducted using a microtube homogenizer (Cole-Parmer), followed by centrifugation and quantification of cleared protein lysate as above.
Organoid samples were digested from reduced growth factor Matrigel in 10mM EDTA in PBS for 1 hour at 4°C, After centrifugation and resuspension in 20–40 μL RIPA buffer, protein lysate samples were cleared and quantified as above.