Ab211522

Cells were fixed with 4% paraformaldehyde, permeabilised with 0.5% Triton X‐100 in PBS for 15 min at room temperature and blocked with 5% BSA for 30 min at 37°C. Cells were treated with primary antibodies at 4°C overnight: ORMDL3 (ab211522, 1:200, Abcam) and p‐PERK (ab192591, 1:200, Abcam). Cells were then incubated with Cy3‐conjugated goat anti‐rabbit (A0516, Beyotime, 1:500) or goat anti‐mouse IgG DyLight 488‐conjugated secondary antibodies (A0428, Beyotime, 1:500) for 2 h at 37°C. Nuclei were stained with DAPI and cells were observed under a fluorescent illumination microscope (Olympus IX71).

Proteins were extracted from tissues or cells in RIPA assay and protein concentration was determined by a BCA protein assays kit (Thermo Scientific). Protein were separated into 10% SDS‐PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp.). The PVDF membranes were incubated by ORMDL3 (ab211522, 1:1000, Abcam), p‐PERK (3179, 1:1000, Cell Signaling Technology, Inc.), PERK (5683, 1:1000, Cell Signaling Technology, Inc.), p‐eiF2α (ab32157, 1:1000, Abcam), eiF2α (ab169528, 1:1000, Abcam), ATF4 (ab184909, 1:1000, Abcam), HSPA5 (ab21685, 1:1000, Abcam), GPX4 (ab125066, 1:1000, Abcam) and β‐actin (ab8226, 1:10000, Abcam) at 4°C overnight. PVDF membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (A0208, A0216, Beyotime, 1:5000) for 2 h. The signal was tested with the chemiluminescence system (Amersham Pharmacia).