Owl semidry electroblotting systems

Protein samples were mixed with SDS sample buffer containing β-mercaptoethanol and denatured at 95°C for 5 min. Proteins were separated on SDS-PAGE gels (Bio-Rad) with constant voltage of 120 V followed by 180 V. SDS-PAGE gels were stained with EZ-Run Protein Gel Staining Solution (Fisher) and de-stained with Milli-Q water. For immunoblot, proteins were transferred onto nitrocellulose membranes (GE Healthcare) at 10 V constant voltage using Owl™ Semidry Electroblotting Systems (Thermo Fisher Scientific). Membranes were incubated with 5% dry milk in 1× TBST (0.05% Tween-20) at RT for 1 h, followed by primary antibody incubation diluted in 0.5% dry milk in 1× TBST (4°C overnight or RT for 2 h). All antibodies used in this study, including their origin and dilutions used for immunoblots are listed in online supplementary Table S2. Blots were washed 3 times with 1× TBST for 20 min each at RT. Membranes were further incubated with diluted secondary antibodies in 0.5% dry milk and 1× TBST at RT for 1 h. Protein signals were developed by Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad) and Amersham™ ECL™ Western Blotting Detection Reagents (GE Healthcare) for alkaline phosphatase-conjugated and HRP-conjugated secondary antibodies, respectively. Images were captured by CCD camera of Azure Biosystems 300 for ECL development.

Protein samples were mixed with SDS sample buffer containing β-mercaptoethanol, and denatured at 95°C for 5 min. Proteins were separated on SDS-PAGE gels (Bio-Rad) with constant voltage at 120V followed by 180V. SDS-PAGE gels were stained with EZ-Run Protein Gel Staining Solution (Fisher) and de-stained with Milli-Q water. For immunoblot, proteins were transferred onto nitrocellulose membranes (GE Healthcare) at 10V constant voltage using Owl^™ Semidry Electroblotting Systems (ThermoFisher Scientific). Membranes were incubated with 5% dry milk in 1xTBST (0.05% Tween-20) at room temperature (RT) for 1 h, followed by primary antibody incubation diluted in 0.5% dry milk in 1xTBST (4°C overnight or RT for 2 h). Blots were washed three times with 1xTBST for 20 min each at RT. Membranes were further incubated with diluted secondary antibodies in 0.5% dry milk 1xTBST at RT for 1 h. Protein signals were developed by Alkaline Phosphate (AP) Conjugate Substrate Kit (Bio-Rad) and Amersham^™ ECL^™ Western Blotting Detection Reagents (GE Healthcare) for AP-conjugated and HRP-conjugated secondary antibodies, respectively. Images were captured by CCD camera of Azure Biosystems 300 for ECL development.