Coimmunoprecipitation Assay for Protein-Protein Interactions

For immunoprecipitation, the following antibodies were used: KDM4B (Cell Signaling; #8639, 1:200 dilution), eIF2α (Santa Cruz; sc-133132, 1:200 dilution), PERK (Cell Signaling; #5683, 1:200 dilution), and P-eIF2α (Cell Signaling; #3398, 1:200 dilution). Coimmunoprecipitation assays were performed as described previously (Wee et al., 2015 (link)) with a modified protocol using NETN buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.3% NP-40) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics) to extract whole-cell protein, or a cell fractionation protocol for cytoplasmic and nuclear fractionation. Cell lysates were incubated with the indicated antibodies at 4°C overnight. The protein complex was captured using protein A–agarose (for rabbit primary antibody) or protein G–agarose (for mouse primary antibody) beads (Roche Diagnostics) at 4°C for 4 h, and agarose beads were collected by centrifuge and washed three times with washing buffer. The precipitated proteins were dissolved in SDS sample buffer along with 3 mM dithiothreitol and subjected to Western blot analysis.