Protein Expression Analysis in Mouse Intestine

To analyze protein expression in the mouse small intestine, the tissue was minced with cold radioimmunoprecipitation assay buffer (RIPA) buffer (Rockland, Limerick, PA, USA), supplemented with 1 mM dithiothteitol (DTT) (Merk, Darmstadt, Germany), and diluted with a phosphatase inhibitor cocktail 2 solution (Merk). After centrifugation, the amount of protein in each supernatant was quantified, mixed with an SDS sample buffer, and denatured for 5 min at 95 °C. Protein electrophoresis and transfer as well as membrane development were all performed as described previously [23 (link),24 ]. Briefly, electrophoresis was performed using 12% Tris-glycine SDS-polyacrylamide gel, and the protein bands were transferred onto a polyvinylidene difluoride membrane, which was blocked for unspecific binding of proteins by incubation at room temperature with 5% skim milk. After three washes with a Tris-Buffered Saline buffer containing 0.1% Tween^® 20, the membrane was incubated with anti-α-defensin-1 or β-Actin antibodies for 3 h, followed by washes and incubation with a secondary antibody. The proteins were detected by using the FUSION Solo Vilber Lourmat system (Collégien, France) with an ECL solution. The intensities of the protein bands were quantified using the ImageJ program and captured images.