Hsp70

Colonic samples (n = 5–6/group per time point) were collected from C57BL/6 female mice receiving 2% DSS or 2% DSS + TUS. Collection was performed at days 0, 3, 5, 7, 9, 11 and 14. After PBS 1× cleaning, samples were snap frozen and processed for further proteomic analysis. Briefly, frozen colonic samples were homogenized in cell lysis buffer (1 mM EDTA, 150 mM NaCl, 0.05% Tween-20 and 20 mM Tris-HCl in ultrapure water) containing Pierce Protease Inhibitor Tablets (Thermo Scientific, Waltham, MA) and 1.0 mm Zirconium Beads. Homogenates were centrifuged at 14,000 rpm at 4 °C for 20 min and the supernatant was collected. The process was repeated two times and aliquots were stored at −80 °C. Bicinchoninic acid assay (BCA – Thermo Scientific, Waltham, MA) was used for protein quantification and samples were further diluted to 1 mg/mL of total protein. MILLIPLEX Map Mouse Cytokine/Chemokine Panel (EMD Millipore, Billerica, MA) was used for proteomic analysis of colonic homogenates according to manufacturer specifications in a Bio-Plex 200 (Bio-Rad Laboratories, Hercules, CA). The same control samples (day 0) were used for multiplex ELISA experiments in the 2% DSS and 2% DSS + TUS exposed mice. Further analysis of colonic TGFβ (Thermo Scientific, Waltham, MA) and HSP70 (R&D Systems, Minneapolis, MN) were performed using ELISA Streptavidin-HRP assay.