pGEX2T-mFOG1(1-45) [15] or pGEX4T1 expression constructs were transformed into an E. coli BL21DE3 strain (C2527H, NEB). The culture was expanded and expression was induced at an OD600 of 0.7 with 0.4 mM IPTG. After 24 h at 18°C bacteria were harvested, washed with PBS and resuspended in PBS/Triton (PBS containing 1% (w/v) Triton X-100). For lysis, cells were sonicated 12 times for 12 s on an ultrasonic homogenizer (HD2200, Bendelin electronics) at 25% output while keeping the suspension on ice in between. The suspension was frozen in liquid nitrogen and thawed on ice three times before cell debris were pelleted by centrifugation at 4°C and 27,000 g for 30 min. GST-fusion proteins were coupled to Glutathione Sepharose 4 Fast Flow (17-5132-01, GE Healthcare) for 2 h at 4°C with rotation. Unbound proteins were removed by washing three times with PBS/Triton and twice with PBS for 5 min at 4°C with rotation. The amount of GST-fusion protein bound to the Sepharose resin was evaluated by comparison to a BSA standard on a Coomassie stained SDS-PA gel.

GST pulldown interaction assays were performed using 10-20 μg of GST fusion proteins and 1 mg of S2 cell nuclear extract or TRAX per pulldown reaction. The resin was blocked for 1 h at 4°C with rotation in GST Pulldown Buffer containing 1 mg/ml BSA and 1% (w/v) fish skin gelatin. Binding took place overnight at 4°C with rotation in 1 ml GST Pulldown buffer (25 mM Hepes/KOH pH 7.6, 150 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 20% (v/v) glycerol 0.1% (w/v) NP-40, 1 mM DTT). The resin was washed four times with 1 ml GST Pulldown buffer for 5 min at 4°C with rotation followed by centrifugation (4 min, 1,500 g, 4°C). Interacting proteins were analyzed by SDS-PAGE and Western blot.

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