Ab230508

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.