Mab1757

Western blot analysis followed published procedures [33 (link)]. In brief, organoids were washed twice in cold PBS, then were cut into small pieces using a razor blade and incubated in RIPA lysis buffer for 30 min on ice. Monolayer cells were washed twice with cold PBS, then incubated in RIPA lysis buffer for 30 min on ice. Lysates were vortexed for 10 sec every 10 min and lastly centrifuged for 30 min at 4 °C and 13,000 rpm. Lysates and media samples were separated by SDS-PAGE and blotted onto PVDF membranes (Bio-Rad, Mississauga, ON, Canada). Blocked membranes were probed with primary antibodies: rat anti-lipocalin-2 (1:500, R&D Systems, MAB1757), rabbit anti-NFκB p65 (1:1000, Abcam, ab16502), mouse anti-STAT3 (1:1000, Abcam, ab119352), mouse anti-β-actin (1:5000, Millipore Sigma, A5316) or mouse anti-alpha-tubulin (1:5000, Abcam, ab7291) overnight at 4 °C. Membranes were washed and incubated with secondary antibodies (goat anti-rat HRP, 1:1000, Thermo Fisher Scientific, 31470, goat anti-rabbit HRP, 1:1000, Bio-Rad, 1706515 and horse anti-mouse HRP, 1:5000, Cell Signaling, Burlington, ON, Canada, 7076S) for 1 h at RT. Membranes were washed and incubated with enhanced chemiluminescence substrate (Bio-Rad) for 5 min, signals were acquired with an Amersham 6000 imager (Amersham, Oakville, ON, Canada) or on film, and quantified in ImageJ.