Anti gpnmb

The whole-cell extracts were lysed in RIPA buffer with a Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The Laemmli’s sample buffer was added to the lysates and boiled at 95 °C for 5 min then analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Western blotting was performed with anti-GPNMB, anti-vimentin, anti-Twist, anti-MMP2 and anti-GAPDH antibodies (Cell Signaling Technology, Danvers, MA, USA). Quantification of protein levels was analyzed by ImageJ software.

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/).