Ab23760

The total protein was extracted by the modified RIPA buffer (P0013B; Beyotime, Shanghai, China), and the concentration was quantitated by the BCA protein assay kit (P0010; Beyotime, Shanghai, China). Then, the total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, and then the membranes were blocked in 5% skim milk diluted with TBST for 1 h at room temperature and probed with primary antibodies, including MFG-E8 (R&D Systems, AF2805, 1 : 500), BCL2 (Abcam, ab117115, 1 : 500), BAX (Abcam, ab216494, 1 : 500), cleaved caspase-3 (Abcam, ab90437, 1 : 500), GRP-78 (Abcam, ab230508, 1 : 500), XBP-1 (Abcam, ab230508, 1 : 500), ATF-6 (Abcam, ab203119, 1 : 500), ATF-4 (Abcam, ab23760, 1 : 500), CHOP (Abcam, ab23760, 1 : 500), p-PI3K (Abcam, ab125633, 1 : 500), PI3K (Abcam, ab154583, 1 : 500), p-AKT (Abcam, ab18785, 1 : 1000), AKT (Abcam, ab28422, 1 : 500), and β-actin (Abcam, ab209857, 1 : 1000) at 4°C overnight. Then, the membranes were incubated with a secondary rabbit anti-rabbit antibody (1 : 1000) the next day at room temperature for 1 h. The ImageJ software was employed for quantitation of the immunoreactive bands.

Paraffin sections of pancreatic tissues from each group were dewaxed, dehydrated with alcohol gradient, washed with tap water for 2 min, treated with H₂O₂ containing 3% methanol for 20 min, washed with distilled water for 2 min, and washed with 0.1 M phosphate buffer saline (PBS) for 3 min followed by antigen retrieval. The sections were added with normal goat serum blocking solution (C-0005, Shanghai Haoran Biological Technology Co., Ltd., Shanghai, China) at 4 °C overnight, added with primary antibody to ATF4 (ab23760, 1:250, Abcam, Cambridge, UK) at 4 °C overnight, and then added with goat anti-rabbit immunoglobulin G (IgG) secondary antibody at 37 °C for 20 min. Next, the sample was added with horseradish peroxidase-labeled streptavidin protein working solution (0343-10000U, Beijing Yimo biological technology Co., Ltd., Beijing, China) at 37 °C for 20 min. After that, the sample was developed by 3,3′-diaminobenzidine (ST033, Guangzhou Weijia technology Co., Ltd., Guangdong, China), stained by hematoxylin (PT001, Shanghai Bogoo Biotechnology Co., Ltd., Shanghai, China) for 1 min, returned to blue color by 1% ammonia, dehydrated by a certain concentration of alcohol, cleared by xylene, mounted by neutral resin and observed under a microscope.

For frozen sections, TM tissues were fixed with 4% paraformaldehyde for 1 h and then dehydrated with a series of sucrose solutions (10–30%) before optimum cutting temperature embedding. Cross sections of the TM were obtained using a cryostat (Leica Biosystems, Buffalo Grove, IL, USA). Sections were immunostained using anti-ATF4 (1:100, #ab23760, Abcam), anti-CHOP (1:100, #sc-575, Santa Cruz Biotechnology), and anti-CD62E (1:500, #550290 from Pharmingen (San Diego, CA, USA) or #S9555 from Sigma-Aldrich) antibodies overnight at 4° C, followed by Cy3-conjugated secondary antibody from Invitrogen, or Alexa Fluor 568 from Thermo Fisher Scientific (Waltham, MA, USA), or followed by biotinylated secondary antibody and fluorescein isothiocyanate avidin. Nuclei were mounted with DAPI-mounting solution. Slides were visualized and photographed under a fluorescent microscope (Axioplan 2 imaging, Carl Zeiss, Heidenheim, Germany).