Western Blotting of ER Stress Proteins

Western blotting was performed as previously described (Watanabe et al., 2022 (link)). Cells were washed with phosphate-buffered saline and lysed in Passive Lysis 5× buffer (Promega, Madison, WI, United States). Nuclear proteins were extracted using a LysoPure Nuclear and Cytoplasmic Extractor Kit (FUJIFILM, Tokyo, Japan). Protein samples were separated on polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with the appropriate primary antibodies, anti-ATF6β (1:1000; 15794-1-AP, Proteintech, Rosemont, IL, United States), anti-RANKL (1:1000; 12A668, Novus Biologicals, United States), anti-CHOP (1:1000, L63F7, Cell Signaling Technology), anti-Grp78 (1:1000, C50B12, Cell Signaling Technology), anti-cleaved caspase-3 (1:1000, 5A1E, Cell Signaling Technology), anti-JNK (1:1000, 56G8, Cell Signaling Technology), anti-Phospho-JNK (Thr183/Tyr185) (1:1000, 81E11, Cell Signaling Technology), anti-Lamin B1 (1:1000, 1298-1-AP, Proteintech) and anti-β-actin (1:1000; 13E5, Cell Signaling Technology) antibodies, and secondary antibodies, anti-rabbit IgG (1:2000; Cell Signaling Technology) and anti-mouse IgG (1:2000; Cell Signaling Technology). Blotted membranes were visualized using a densitometry technique on Amersham ImageQuant 800 (Cytiva, Tokyo, Japan) and quantified using Multi Gauge 3.1 software (FUJIFILM, Tokyo, Japan).