Can get signal 2

Western blotting was performed as described previously (39 (link)). Briefly, cells were lysed with NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris–Cl pH 8.0) with Complete Protease Inhibitor Cocktail (Sigma-Aldrich, MO), and sonicated using a Bioruptor (SonicBio, Kanagawa, Japan) at high power, on a 30 s-off 30 s-on cycle, 4 times. SDS-PAGE was performed using a 5–12% polyacrylamide gel (FUJIFILM Wako, Osaka, Japan) and NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific) for 1 h, 120 V. One microgram of protein was loaded into each lane. After electrophoresis, proteins were transferred to Immobilon PVDF membranes (Merck, Darmstadt, Germany) in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol) using Transblot SD cell (Bio-Rad, CA). After blocking the membranes with SuperBlock Blocking Buffer (PBS) (Thermo Fisher Scientific) for 30 min, they were soaked in Can Get Signal 1 with primary antibodies and incubated overnight at 4°C. After three washes with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, 0.1% Tween 20), the membranes were incubated with secondary antibodies prepared in Can Get Signal 2 (TOYOBO) for 1 h at 20–25°C. After a triple wash with PBST, the signals were detected using Immunostar LD (Fujifilm Wako, Osaka, Japan) and ChemiStage CC-16 Mini (KURABO, Osaka, Japan). Antibodies are shown in Supplementary Table S3.

Immunoblotting was performed as previously described [22 (link), 31 (link)]. Cells were collected, washed with PBS and lysed in RIPA buffer (1% Triton X‐100, 0.5% deoxycholate, 0.1% SDS, 150 mm NaCl and 50 mm Tris/HCl at pH 8.0) containing 1× cOmplete Protease Inhibitor Cocktail (Roche Diagnostics). Cytochrome release was measured using a Cytochrome c Release Assay Kit (Abcam) according to the manufacturer’s instructions, subjected to SDS/PAGE, and transferred onto polyvinyl fluoride membranes (Pall Corporation, East Hills, NY, USA) blocked with 5% skim milk containing Tris‐buffered saline with Tween20 (Sigma‐Aldrich; TBST). The membranes were incubated with primary antibodies and the appropriate secondary antibodies (Table S3) diluted in TBST, Can Get Signal 1 (Toyobo, Osaka, Japan) or Can Get Signal 2 (Toyobo). Chemiluminescence images were captured using an ImageQuant LAS 4000 device (Fuji Film, Tokyo, Japan).