Immunoblot analysis of intestinal proteins

Protein was extracted from intestinal mucosa dissected from mouse intestine (20–50 µg protein per lane) or total lysates from cells (10⁶/well, 6-well plates) from in vitro experiments. Tissue or cell lysates were extracted in T-Per or M-Per (Thermo Fisher Scientific), respectively, supplemented with protease and phosphatase inhibitors (Roche, St. Louis, MO). Protein was quantified by immunoblot analysis employing antibodies to: GFP (cat. #13970, 1:1000), Axin2 (cat. #32197, 1:1000), c-Myc (cat. #32072, 1:1000) from Abcam; GAPDH (cat. #2118, 1:5000), β-catenin (cat. #8480, 1:1000), human APC (cat. #2504, 1:1000), TCF4 (cat. #2569, 1:1000), β-actin (cat. #4967, 1:3000), phosphorylated VASP-ser239 (cat. #3114, 1:1000) from Cell Signaling Technology (Danvers, MA); mouse APC (cat. #896, 1:1000) from Santa Cruz Biotechnology (Dallas, TX); GUCA2A (cat. # HPA018215, 1:100) from Sigma or guanylin antisera (#2538, 1:100);^{56 (link)} and GUCY2C (1:500).^{34 (link)} Immunoblot images were captured on the BioRad ChemiDoc MP imaging station and bands were quantified by densitometry normalized to that of GAPDH or β-actin using ImageJ. Average relative intensity reflects at least two independent experiments each with at least three biological replicates.