1,500 organoids treated or not with 100 nM 4OT for 7 d were harvested using 1 ml Corning Cell Recovery Solution (354253; Corning) per ∼60 µl BME gel. After centrifugation, they were lysed in 100 µl RIPA buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 5 mM EDTA, 50 mM NaF, and 1 mM PMSF supplemented with Roche 1X Halt Protease inhibitor cocktail), incubated for 1 h at 4°C, and sonicated using a Bioruptor PLUS (Diagenode) for 30 cycles of 30 s spaced by 30 s of cooling down. Total protein concentration in lysates was determined using Pierce BCA Protein Assay Kit (23227; Thermo Scientific). The protein concentration in the lysates was 1.74 µg/µl. Proteins were fractionated in 10% polyacrylamide gel and blotted on polyvinylidene difluoride membrane. Milk was used as a blocking agent.
Membranes were incubated with a Rabbit anti GORASP2 (Rich, against aa 232–454), at dilution 1/1,000; a gift from M. Bekier (University of Michigan, Ann Arbor, MI; Xiang and Wang, 2010 (link)); and a mouse monoclonal antibody to E-cadherin (610182 at 1/5,000; BD Biosciences).
The visualization was performed after incubation with secondary antibodies coupled to HRP (NA931 and NA934; GE Healthcare) and Clarity Western ECL Substrate (Bio-Rad) using ImageQuant LAS4000 ECL (GE Healthcare).