Flag resin

For expression in mammalian cells constructs of DNTTIP1, MIDEAS and HDAC1 were cloned into the pcDNA3 vector. The FLAG tagged constructs contained an N-terminal 10xHis-3xFLAG tag and a TEV protease cleavage site. HEK293F cells (Invitrogen) were co-transfected with mixtures of both tagged and untagged constructs using polyethylenimine (PEI) (Sigma). To transfect cells, 30 μg DNA total was diluted in 3 ml of PBS (Sigma) and vortexed briefly; 120 μl of 0.5 mg/ml PEI was added, and the suspension was vortexed briefly, incubated for 20 min at room temperature, then added to 30 ml cells (final density was 1 × 10⁶ cells/ml). Cells were harvested 48 h after transfection. For the interaction studies, the cells were lysed by sonication in buffer containing 50 mM Tris/Cl pH 7.5, 100 mM potassium acetate, 5% (v/v) glycerol, 0.3% (v/v) Triton X-100 and Complete EDTA-free protease inhibitor (Roche) (buffer A); the insoluble material was removed by centrifugation. The complex was then bound to FLAG resin (Sigma), washed three times with buffer A, three times with buffer B (50 mM Tris/Cl pH 7.5, 300 mM potassium acetate, 5% (v/v) glycerol) and three times with buffer C (50 mM Tris/Cl pH 7.5, 50 mM potassium acetate, 5% (v/v) glycerol, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP)). The complex was eluted from the resin by overnight cleavage at 4°C with TEV protease in buffer C.

The HDAC1/MIDEAS/DNTTIP1 complexes were purified from 1.2 l of HEK293F cells expressed as described above but with 300 ml of cells in 2 l roller bottles. The cells were lysed by sonication in buffer containing 50 mM Tris/Cl pH 7.5, 100 mM potassium acetate, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100, and Complete EDTA-free protease inhibitor (Roche) (buffer A); the insoluble material was removed by centrifugation. The complex was then bound to FLAG resin (Sigma), washed twice with buffer A, three times with buffer B (50 mM Tris/Cl pH 7.5, 50 mM potassium acetate, 5% (v/v) glycerol, 0.5 mM TCEP), incubated with 0.5 mg RNaseA for 1 h at 4°C and then washed five times with buffer B. The complex was eluted from the resin by overnight cleavage at 4°C with TEV protease in buffer B. The complex was further purified by gel filtration on a Superdex-200 column (GE Healthcare).

HEK293T/17 (1.5 × 10⁶ cells/10 cm plate) were transfected for 24 hours, and whole cell extracts were prepared as described [51 (link)]. Proteins were then immunoprecipitated from 200–400 μg of whole cell extracts as described [93 (link)] and analyzed by Western blot. For immunoprecipitation of endogenous HBO1, HBZ-Flag was transfected into H1299 cells for 48 hours, nuclear extracts were then prepared and HBZ was immunoprecipitated with Flag-resin (A2220, Sigma-Aldrich).

The antibodies used in our study were as follows: anti-FLAG M2 monoclonal (Sigma, St. Louis, MO, USA), anti-NRF2 (Abcam, Cambridge, UnitedKingdom), anti-β-actin (Cell Signaling,Danvers, MA, USA), anti-HO-1 (Cell Signaling), anti-LaminB1 (Cell Signaling), anti-Lamin A/C (Cell Signaling). The Nuclear and Cytoplasmic Protein Extraction kit was obtained from (Thermo Fisher Scientific, Waltham, MA, USA). For immunoprecipitation, Whole cell lysate (WCL) was used respectively as the negative control. Cell lysates were cleared by centrifugation and were incubated with FLAG resin (Sigma) before washing with lysis buffer, followed by overnight incubation at 4 °C. After washing three times by 1× phosphate-buffered saline (PBS), the precipitates were analyzed by immunoblotting.