Fastprep bead beater system mp bio

Protein was isolated from intestinal tissue segments of ~1cm in 500 μl RIPA buffer with protease inhibitors with the Fastprep bead-beater system (MP Bio, BioSpec). Samples were subjected to 4 rounds of lysis at speed 6 for 20 seconds at 4°C. Primary intestinal epithelial cells were lysed in Transwells with 50 μl RIPA buffer with protease inhibitors (Sigma). Total protein was quantified by Pierce BCA Protein Assay Kit (Thermo Scientific). Supernatants from fecal samples and Transwells were taken as described above. Samples were run on SDS- PAGE gels (AnykD or 7.5% Mini-Protean TGX gels, Bio Rad) and transferred onto nitrocellulose membrane (Bio Tris-Rad). Membranes were blocked with 3% milk in 0.1% Tween-20 tris-buffered saline for 1 h at room temperature and probed with goat IgG anti-pIgR/SC (R&D, catalog #AF2800) and rabbit IgG anti-Actin (Sigma, catalog #A2066) overnight at 4°C. Blots were incubated for 1 h with horseradish peroxidase conjugated secondary antibodies (Invitrogen catalog #A16005, BioRad catalog #170-6515) before development with the SuperSignal West Dura chemiluminescent kit (Thermo Scientific). Immunoblots were quantified by ImageJ software^{35 (link)}. Whole tissue pIgR/SC was normalized to actin, fecal samples were normalized by weight as described above, and apical supernatants were normalized by volume.