Anti lactoferrin

Milk-exo and Exo-free milk were quantitated by bicinchoninic acid (BCA) protein assays, and the proteins were separated with 8–12% sodium dodecyl sulfate polyacrylamide gel and transferred to nitrocellulose membrane using a Trans-Blot Turbo (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% skim milk for 1 h at room temperature followed by incubating with primary antibodies at 4°C overnight: anti-Tsg101 (1:1000, Abcam, Cambridge, MA, USA), anti-Alix (1:500, Novus Biologicals, Littleton, CO, USA), anti-MFG-E8 (1:1000, R&D Systems, Wiesbaden-Nordenstedt, Germany), anti-GM130 (1:1000, Invitrogen™ Thermo Fisher Scientific, Waltham, MA, USA), anti-Lactoferrin (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Wnt3a (1:1000, Abcam), anti-β-catenin (1:500, Abcam), GAPDH (1:1000, R&D Systems). After 3 times wash with TBST (Tris-buffered saline, 0.1% Tween 20), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The protein bands were visualized with iBright™ CL750 Imaging System (Invitrogen™ Thermo Fisher Scientific, Waltham, MA, USA).