Phosstop

Organoids from the same condition were pooled (Total 6) in an Eppendorf and rinsed with PBS. The PBS was removed, and the organoids were frozen at -80°C before extraction. Radio immunoprecipitation assay (RIPA) lysis buffer (ref. 89901, ThermoScientific) supplemented with Phosstop (ref. 04 906 837 001, Roche) and cOmplete tablets (ref. 4693116001) were used to lyse the organoids under sonication to perform protein extraction. The amount of protein was quantified using the DC protein assay kit (ref. 500-0116, Bio-Rad) according to the supplier’s recommendations.

Protein extracts were obtained by lysing cells in TNN buffer supplemented with 1× Complete ULTRA protease inhibitor (Roche, Basel, Switzerland) and 1× PhosSTOP (ThermoFisher Scientific, Waltham, MA, USA). A total of 25 µg protein was separated on 4–10% Criterion TGX Stain-Free Protein Gels (BioRad, Hercules, CA, USA) or Mini-PROTEAN TGX Stain-Free Protein Gels (BioRad) and immediately transferred to Trans-Blot Turbo Midi PVDF Transfer Packs (BioRad) or Trans-Blot Turbo Mini Transfer Packs (BioRad). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at RT followed by incubation with anti-human CDKN2A antibody (Abcam, ab108349, 1:1000), anti-human Mesothelin antibody (Abcam, ab93620, 1:150), anti-human Podoplanin antibody (Abcam, ab236529, 1:500), and anti-human NF2 antibody (Abcam, ab109244, 1:10,000) overnight at 4 °C. The detection antibody HRP anti-rabbit IgG (BioLegend, San Diego, CA, USA, 410406, 1:1000) was incubated for 1 h at RT. Membranes were imaged using either Clarity Western ECL Substrate (Biorad) or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) on a Fusion FX (Vilber Lourmat, Eberhardzell Germany). GAPDH and α-Tubulin were used as loading controls, anti-human GAPDH antibody (ThermoFisher Scientific, MA5-15738, 1:1000), and anti-human α-Tubulin antibody (Sigma-Aldrich, T5168, 1:1000).

Lysates of mice skeletal muscle tissue generated under the addition of proteinase inhibit cocktail Complete Mini (Thermo, Waltham, MA, USA) and phosphatase inhibitor cocktail PhosSTOP (Thermo, Waltham, MA, USA). The total protein of the lysates was measured by the Pierce BCA Protein Assay Kit (Thermo, Waltham, MA, USA). Co-immunoprecipitation (co-IP) was completed using the Thermo Scientific Pierce co-IP kit (#26149) following the manufacturer’s protocol. Ten micrograms of the antibody were incubated with the delivered resin and covalently coupled. The antibody-coupled resin was incubated with 200 µL of the whole mice skeletal muscle protein lysates overnight at 4 °C, respectively. The resin was washed, and the protein complexes bound to the antibody were eluted. Subsequent Western blot analyses were performed as described before.