Antihuman pro caspase 3

Immunoprecipitation experiments were performed using an amine-reactive resin-based method (Pierce, #26149), using antihuman p21^CIP1 (Novus Biologicals, #AF1047, 1:200). Immunoblotting was performed using the antibody antihuman pro-Caspase 3 from ThermoFisher Scientific (#31A893, 1:300). Primary antibodies were detected with peroxidase-conjugated antirabbit or anti-mouse IgG (1:3000) and visualized with SuperSignal West Femto Substrate (ThermoFisher Scientific #34094).

In vitro and grafted organoids were lysed in RIPA buffer containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo FisherScientific), using a glass Dounce homogenizer (DWK Life Sciences Kimble™) on ice. Following protein quantification, SDS-PAGE gel run and western blotting were performed. The following antibodies from Cell Signaling Technology were used: antiribosomal protein S6 (#2217, clone 5G10, 1:1000), antiphospho S6 (#2211, 1:1000), antiphospho P70S6K (#9205, 1:1000), anti-P70S6K (#9202, 1:2000), anti-β Actin (#4967, 1:1000), anti-GPNMB (#38313, clone E4D7P, 1:700). Anti-ACTA2 was from Sigma-Aldrich (#F3777, clone 1A4, 1:2000), antihuman pro-Caspase 3 was purchased from ThermoFisher Scientific (#MA1-41163, clone 31A893, 1:400), antihuman cleaved Caspase 3 was purchased from Abcam (ab2302, 1:200), antihuman p21CIP1 was purchased from Novus Biologicals (#AF1047, 1:400). Primary antibodies were detected with peroxidase-conjugated antirabbit or anti-mouse IgG (1:3000) and visualized with SuperSignal West Femto Substrate (ThermoFisher Scientific #34094). Blots were quantified using ImageJ v1.51 W (https://imagej.nih.gov/ij/).