Immunoblot analysis was performed based on our previous methods (Eguchi et al., 2018 (link); Sakurai and Kuwahara, 2021 (link)). Cells were washed with PBS on ice and lysed in a lysis buffer containing 50 mM Tris HCl, pH 7.6, 150 mM NaCl, 0.5% (vol/vol) Triton X-100, Complete EDTA-free protease inhibitor cocktail 3 (Roche), and PhosSTOP phosphatase inhibitor Cocktail (Roche). Lysates were centrifuged at 20,800 × g for 5 min at 4°C and supernatants were mixed with NuPAGE LDS Sample Buffer (4 ×) buffer (Thermo Fisher Scientific). For SDS-PAGE, samples were loaded onto Tris-glycine gels and electrophoresed. After electrophoresis, samples were transferred to polyvinylidene fluoride membranes. Transferred membranes were blocked and incubated with primary antibodies and then with HRP-conjugated secondary antibodies (Jackson ImmunoResearch). Protein bands were detected by LAS-4000 (FUJIFILM). The integrated densities of protein bands were calculated using ImageJ software.
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