Western Blot Immunodetection Protocol

Immunoblotting was performed as described previously^{14 (link)}. Briefly, cell lysates were made in lysis buffer (pH 7.5) containing 1 mM EDTA, 50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium vanadate, and 1 μg/ml leupeptin, aprotinin, and pepstatin. Proteins were then separated by SDS-PAGE gels and further transferred to the membranes of polyvinylidene difluoride (Bio-Rad, Hercules, CA, USA). After blocking the membranes with 5% nonfat milk for 1 h at room temperature, they were incubated with primary antibodies as indicated. The membranes were then washed and incubated at room temperature with peroxidase-conjugated secondary antibodies for 1 h. After 5 times of washing, immunodetected bands on the membranes were visualized by taking chemiluminescent images on X-ray films with the enhanced chemiluminescence (ECL) system (PerkinElmer, Waltham, MA, USA) as per the manufacturer’s instructions. All uncropped scans are shown in Supplementary Fig. 13 for the western blots.