Supersignal west femto maximum sensitivity chemiluminescence substrate

Protein extracts from whole cell lysates containing ∼ 40 μg protein were loaded in each lane of a Mini-Gel module for electrophoresis (BioRad, Munich, Germany). Protein was transferred onto nitrocellulose membrane (Amersham Protran 0.45 μm NC Western Blotting Membrane, GE Healthcare, USA), blocked with 1x Blocking buffer (Pierce™ Protein-Free (TBS) Blocking Buffer, Thermo Fisher) at RT for 1 h, and incubated in primary antibody at 4 °C overnight. GAPDH (Rabbit Anti-GAPDH (D16H11) mAb, Cell Signaling Technology) was used as the loading control at 1:1000 dilution. Blots were incubated with secondary antibody at 1:10000 dilution in blocking buffer for 1 h at RT. Protein bands were visualized with SuperSignal™ West Femto Maximum Sensitivity Chemiluminescence Substrate (Thermo Scientific). Densitometric intensities were calculated using the ImageLab software (BioRad). Primary antibodies are listed in supplementary experimental procedures.

Protein extracts from whole cell lysates containing ∼40 μg protein was loaded in each lane of a Mini-Gel module for electrophoresis (Bio-Rad, Munich, Germany). Protein was transferred onto nitrocellulose membrane (Amersham Protran 0.45 μm NC Western Blotting Membrane, GE Healthcare, USA), blocked with 1x blocking buffer (Pierce™ Protein-Free (TBS) Blocking Buffer, Thermo Fisher, Waltham, MA, USA) at room temperature (RT) for 1 h, and incubated in primary antibody at 4°C overnight. GAPDH (Rabbit Anti-GAPDH (D16H11) mAb, Cell Signaling Technology, Danvers, MA, USA) was used as the loading control at a 1 : 1000 dilution. Blots were incubated with the secondary antibody at a 1 : 10000 dilution in blocking buffer for 1 hour at RT. Protein bands were visualized with a SuperSignal™ West Femto Maximum Sensitivity Chemiluminescence Substrate (Thermo Scientific). Densitometric intensities were calculated using Image Lab software (Bio-Rad). Primary antibodies are listed in Table 1.