Chemodoc mp imaging system

Proteins were transferred to a 0.45-μm nitrocellulose membrane (Millipore, IPVH00010) and then blocked with 3% bovine serum albumin in Tris-buffered saline with Tween-20 (TBST, 20 mM Tris-HCl, pH 7.4, 137 mM NaCl and 0.1% Tween 20) at room temperature for 1 h. Membranes were then incubated with anti-Flag antibody (ABclonal, AE005, 1:3000) or anti-HA antibody (ABclonal, AE008, 1:3000) at 4 °C overnight. The membranes were washed three times with TBST buffer, incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (ABclonal, AS003, 1:4000), and visualized on a ChemoDoc MP imaging system (BIO-RAD).

Cells were lysed in CelLytic buffer (Sigma) supplemented with 1% protease inhibitor ‘cocktail’ (Pierce). Protein samples were subsequently separated by electrophoresis through NuPAGE (Invitrogen) 4–12% Bis-Tris gradient gels or 4–12% pre-cast XT-Criterion gradient gels (Biorad, Hercules, CA), and were then transferred to PVDF membranes (Immobilion; Millipore). Afterwards, blots were blocked in 5% milk in TBST and incubated overnight at 4 °C with primary antibodies (Supplementary Table 4). After incubation with primary antibodies, blots were washed with 0.1% TBST three times for 10 minutes each, followed by incubation with secondary antibodies (goat anti-mouse or goat anti-rabbit antibodies, ThermoFisher Scientific) conjugated with alkaline phosphatase (AP) in 5% milk in TBST at room temperature for an hour. Washing was repeated as above, and blots were visualized using Novex™ AP Chemiluminescent Substrate (CDP-Star™) (ThermoFisher Scientific). Blots were developed either on films or on Biorad Chemodoc MP Imaging system with Image Lab 5.1 software.