Western blot was performed using Anti-TFF1 Polyclonal Antibody (Product # PA5-80108) and a 9kDa band corresponding to Trefoil factor 1 was observed in MCF7 cell line that also showed upregulation upon treatment with beta-estradiol as opposed to MDA-MB-231 or MCF 10A. Relatively high expression was also observed in Mouse Stomach tissue lysate as compared to Mouse Testis as reported in the literature. Whole-cell extracts (30 µg lysate) of MCF7 (Lane 1), MCF7 treated with beta-estradiol (10 nanomolar for 24h) ( (Lane 2), MCF7 (Lane 3), MCF7 treated with PTI (1X for 4h) (Lane 4), MDA-MB-231 (Lane 5), MDA-MB-231 treated with PTI (1X for 4h) (Lane 6), Mouse Stomach (Lane 7), Mouse Testis (Lane 8), MCF 10A (Lane 9), MCF 10A treated with PTI (1X for 4h) (Lane 10) were electrophoresed using Novex™ 16% Tricine Protein Gel (Product # EC6695BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL concentration) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).