Ero1a

Immunohistochemistry (IHC) staining was routinely carried out using an immunohistochemistry kit (abcam, UK). The tissue sections were deparaffinized and rehydrated. The antigen was retrieved by sodium citrate buffer. Following endogenous peroxidase blocking and incubation in normal goat serum (Vector Lab, Burlingame, CA) for 20 mins, these sections were incubated with ERO1A (1:200, LSBio, USA), CEA (Use directly, zsbio, China), CA19-9 (Use directly, zsbio, China) overnight at 4°C and then with the peroxidase-conjugated secondary antibody (abcam, UK). Color development was performed with 3,3′-diaminobenzidine (DAB) and slides were counterstained with hematoxylin. Neutral resin sealing was performed. The results of immunohistochemical staining were observed under a microscope (Olympus Tokyo, Japan).

Total proteins were extracted from cultured cells using pierce T-PER protein extraction reagent (cwbiotech, China) and a sonicator (SONICS, USA). Protein quantification with BCA (Beyotime, Shanghai, China) Kit. Samples were denatured in sodium dodecyl sulfate (SDS) sample buffer. Total proteins were separated by loading 40 μg of total cell lysate onto SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies against ERO1A (1:1,000, LSBio, USA), actin (1:3,000, ABCAm, USA), p-Akt (1:1,000, CST, USA), Akt (1:1,000, CST, USA), p-4EBP1 (1:1,000, CST, USA), 4EBP1 (1:1,000, CST, USA), p-S6 (1:1,000, CST, USA), S6 (1:1,000, CST, USA), E-cadherin (1:1,000, CST, USA), Vimentin (1:1,000, CST, USA), Snail (1:1,000, CST, USA), Slug (1:1,000, CST, USA), MMP-2 (1:1,000, CST, USA) and MMP-9 (1:1,000, CST, USA), and enzymatic signals were visualized using a chemiluminescence kit (Beyotime Biotechnology, China).