Western blocking reagent

Proteins were separated by SDS-PAGE using NuPAGE 4%-to-12% (wt/vol) Bis-Tris gradient gels (Thermo Fisher Scientific) and were then transferred onto a polyvinylidene difluoride (PVDF) membrane using an iBlot 2 dry blotting system (Thermo Fisher Scientific). To detect biotinylated proteins, 5 μg of total protein and 20 μl of purified protein were loaded and membranes were blocked with Western blocking reagent (Roche) and then incubated with Pierce streptavidin-horseradish peroxidase (HRP) (Thermo Fisher Scientific, catalog no. f21130) (1:2,000) overnight at 4°C. To detect the Myc-tagged proteins, a 10-μg volume of total protein was loaded and membranes were blocked with a 5% (wt/vol) solution of skim milk and then incubated with primary antibody (Myc-tagged mouse monoclonal antibody [MAb]; Cell Signaling Technology, catalog no. f2276) (1:1,000) overnight at 4°C. Hybridization with a secondary antibody (HRP-linked anti-mouse IgG; Cell Signaling Technology, catalog no. f7076) was performed for 1 h at room temperature. Chemiluminescence of HRP substrate was detected with a Fusion FX7 system (Vilber Lourmat, Germany).

Protein was extracted using RIPA buffer (Thermo Fisher) supplemented with protease inhibitor cocktail (Roche) followed by boiling in 1× LDS sample buffer and 1× reducing reagent (both Thermo Fisher) for 5 min at 95°C. 30, 15 or 7.5 μg of protein was loaded per sample and run in 4–12% Bis–Tris gels (Thermo Fisher) in 1× MOPS or MES buffer (Thermo Fisher). Dry blotting was performed using an iBlot™ device (Thermo Fisher) and PVDF iBlot™ gel transfer stacks. Membranes were blocked in 0.1% Tween-20 in PBS with 1:10 dilution of western blocking reagent (Thermo Fisher) for a minimum of 45 min at room temperature followed by primary antibody incubation at 4°C overnight and secondary antibody incubation for 1 h at room temperature (for antibodies and dilutions see; supplementary information, Table S2). HRP stained western blots were developed using SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific) and imaged using an ImageQuant LAS 4000 (GE Healthcare). Near infrared stained blots were imaged on an Odyssey Fc system (LI-COR, Nebraska, USA).