Np0326box

Constructs were washed in PBS for 3 times and then pulverized in RIPA buffer (Sigma Aldrich) supplemented with 1% (v/v) protease and phosphatase inhibitor cocktail (ThermoFisher). After centrifugation, protein concentrations of the supernatants were determined using the BCA Protein Assay Kit (Thermo Scientific BCA Protein Assay Kit). After thermal denaturation and reduction in Laemmli buffer containing β-mercaptoethanol (10% (v/v) (BioRad; Hercules, California), the proteins were electrophoretically fractionated in a 4–12% Bis-Tris polyacrylamide gel (Invitrogen™, NP0326BOX) and transferred onto PVDF membranes (0.2 μm). After being blocked in 3% non-fat milk (BioRad) in TBST (0.1% Tween-20 in TBS) for 1.5 h at room temperature, the membranes were incubated at 4°C overnight with primary antibodies against target proteins (diluted in 1% non-fat milk in TBST; see information in Table S2). After washing in TBST for 5 times, the membranes were incubated with horseradich peroxidase (HRP)-conjugated secondary antibodies (GENA934-1ML, Sigma-Aldrich, 1:2000 dilution) for 1.5 h at room temperature and then with SuperSignal West Femto Maximum Sensitivity Substrate solution (ThermoFisher). The blots were imaged using the ChemiDocTM Touch Imaging System (Bio-Rad). Quantification of the blot images was conducted using NIH ImageJ.