Protein Extraction and Western Blot Analysis

Proteins were extracted from the jejunum and ileum samples and IPEC-J2 cells, as previously described (Zhang et al., 2015 (link); Yang et al., 2017 (link)). The primary antibodies were rabbit anti-phospho-Ser473 (p)-Akt (1:1000, ab138726), rabbit anti-phospho-Tyr1068 (p)-EGFR (1:250, ab32430; Epitomics, Burlingame, CA), rabbit anti-LC3A/B (1:1000, Cell Signaling Technology, Danvers, MA), rabbit anti-total-EGFR (1:500, 18986-1-AP), rabbit anti-total-Akt (1:1000, 10176-2-AP), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:500, 60004-1-Ig, Proteintech Group, Chicago, IL). Horseradish peroxidase-conjugated AffiniPure goat anti-mouse IgG (1:5000, SA00001-1; Proteintech Group) or goat anti-rabbit IgG (1:5000, SA00001-2; Proteintech Group) were used as secondary antibodies.