Intestinal Mucosa Protein Analysis

Intestinal mucosa samples were homogenized with lysis buffer for 30 seconds in a mortar and pestle with liquid nitrogen. Homogenates were centrifuged at 13000 rpm for 10 min at 4°C and the supernatant was collected as the source of protein sample. The proteins were processed with standard methods for Western blot analysis as described [10 (link)]. Rat monoclonal anti-tryptase antibody, rat monoclonal anti-gp91^phox and anti-p47^phox antibodies, rat monoclonal anit-P-selectin and anti-ICAM-1 antibodies, and α-tubulin antibody were obtained from Santa Cruz (Santa Cruz, CA, USA). The secondary antibody conjugated to horseradish peroxidase was diluted at 1 : 2,000 (Santa Cruz, USA). Immunoblots were incubated with an enhanced chemiluminescence detection system (KeyGen Biotech, China) and the densitometry analysis was performed using Quantity One software.