Protease and phosphatase

Scholars extracted the total proteins for 60 min on ice in RIPA buffer (Thermo Scientific, USA) having protease and phosphatase as an inhibitor (Cell Signaling Technology, USA). Cell lysates were centrifuged at 1.2 × 10⁴ g, 4°C for 15 min, and the concentrations of supernatants were examined with a BCA Protein assay kit (Thermo Scientific, USA). 30 μg protein was separated by 10% SDS-PAGE (Life Technology, USA) and then moved to 0.45 μm PVDF membranes (Millipore, USA). After that, the PVDF membranes were instantaneously obstructed by 5% defatted milk in 1 × tris-buffered saline (pH 7.6, 50 mM Tris and 150 mM NaCl) for about 2 h at a regular temperature. The membranes were incubated with monoclonal antibodies at 4°C for 24 h. In total, primary antibodies included those for YWHAB (Abcam, UK, ab32560), NOL10 (Abcam, UK, ab181161), PPAT (Abcam, UK, ab125864), GAPDH (Abcam, UK, ab8245). We washed membranes for three times then incubated with fluorescent-tagged secondary antibody for another 2 h. Then, we used electrochemiluminescence (ECL) to read and the immunoblots were examined with a visible imaging system (Bio-Rad, USA).