Cell lysates were prepared by resuspending cells from a 10 cm2 culture well in lysis buffer (50 mM Tris-HCl/50 mM 3-(N-Morpholino)propanesulfonic acid [MOPS] [pH 7.7], 0.1% SDS, and 1 mM EDTA) supplied with protease inhibitors (Roche) and benzonase (New England Biolabs), followed by 30 min incubation at 4°C and filtration with a 0.22 μm spin filter (Millipore). Conditioned medium was centrifuged for 5 min at 500 × g, and the cell pellet was discarded. The supernatant was filtered using a 0.22 μm syringe filter or centrifuged at 4°C at 18,000 × g for 15 min.
Proteins separated by SDS-PAGE were detected with SimplyBlue SafeStain (Invitrogen/Thermo Fisher Scientific) or transferred to nitrocellulose membranes (GE Healthcare) for immunoblotting with Penta·His mouse monoclonal antibody (1:1,000, QIAGEN) or 9E10 anti-Myc mouse monoclonal antibody (1:2,000, Sigma-Aldrich). Chemiluminescence detection was performed with Western Lightning ECL Plus (PerkinElmer).
Proteins separated by SDS-PAGE were detected with SimplyBlue SafeStain (Invitrogen/Thermo Fisher Scientific) or transferred to nitrocellulose membranes (GE Healthcare) for immunoblotting with Penta·His mouse monoclonal antibody (1:1,000, QIAGEN) or 9E10 anti-Myc mouse monoclonal antibody (1:2,000, Sigma-Aldrich). Chemiluminescence detection was performed with Western Lightning ECL Plus (PerkinElmer).