Western Blot Analysis of Mucin Proteins

The immunodetection was carried out according to previous reports^{38 (link)}. Briefly, nitrocellulose membranes were blocked in 5% (w/v) milk in 1X Tris buffered saline-Tween (TBST) for 1 h at room temperature. Membranes were then washed in TBST and incubated at room temperature overnight with the MUC2 rabbit polyclonal antiserum, MAN-2I, the MUC5AC rabbit polyclonal antiserum, MAN-5ACI, the MUC5B monoclonal antibody, EUMUC5B, and the mouse anti-6X His-tag monoclonal antibody [Cat# ab15149, RRID:AB_301694; Abcam, MA, USA] (for His-tagged MUC5B protein domains, NT5B and CT5B) all at a dilution of 1:2,000 in 1X TBST. Blots were then washed and incubated with fluorophore-conjugated secondary antibody [IRDye 800 goat anti-mouse (Cat# 926-32210, RRID:AB_621842) or IRDye 800 goat anti-rabbit (Cat# 926-32211, RRID:AB_621843); LI-COR Biosciences, Cambridge, UK] at a dilution of 1:25,000 in TBST for 60 min. Finally, each blot was imaged using a LI- COR Odyssey® CLx Infrared Imaging System.