SBP-pulldown

293TT cells were transfected with an equal amount of each expression plasmid using PEI, and the cells were cultured for 24 h. Then, the cells were washed twice with PBS and suspended in T lysis/wash buffer. The cell lysate was sonicated and centrifuged at 20,000 × g for 30 min at 4 °C. The supernatant was used as the input for an SBP-pulldown assay. For the assay, an appropriate amount of Streptavidin Mag Sepharose was washed three times with T lysis/wash buffer and blocked with 0.1% BSA in PBS at 37 °C for 1 h. After the three washes, the Sepharose was incubated with the supernatant at 4 °C overnight with gentle agitation. Then, the Sepharose was washed with T lysis/wash buffer five times (5 min per wash), and the Sepharose-binding proteins were eluted with 1 × SDS loading buffer (50 mM Tris–Cl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 0.1% bromophenol blue, 10% glycerol, and 100 mM dithiothreitol [DTT]) and incubated at 95 °C for 10 min. This sample was used for western blot analysis.