1xprotease inhibitors cocktail

HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with fetal bovine serum (10%, Gibco, USA), penicillin (100 µg ml⁻¹) and streptomycin (100 mg ml⁻¹, Sangon Biotech), under conditions of 5% CO₂ at 37°C. Cells were subcultured for further experiments after they became 80%–90% confluent. The transfection was performed according to the manufacturer's instructions using Lipofectamine 3000 Invitrogen (Thermo Fisher).
After transfection (48 h), the cells were collected and lysed in buffer containing 50 mM Tris–HCl (pH8), 75 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 100 mM sucrose, 1 mM DTT and 1xProtease Cocktail inhibitors (Roche) for 30 min at 4°C. Lysates were centrifuged for 10 min at 14 000 rpm to remove debris. The supernatant was incubated with GFP-tap beads (gta 20, chromotek, Germany) and incubated on a rotator overnight at 4°C. The immunoprecipitants were washed with 1 ml wash-buffer containing 10 mM Tris–HCl (pH7.5), 150 mM NaCl, 0.5 Mm EDTA, 1 mM DTT and 1xProtease inhibitors Cocktail (Roche) for three times. The immunoprecipitants were eluted using 2× SDS-PAGE Sample Buffer and boiled for 10 min at 95°C. Samples were loaded into 10% SDS-PAGE gels and electrophoretically separated.

For Western blotting, following appropriate treatment where applicable, cells were lysed in Hepes lysis buffer [100 mmol/l NaCl, 1% (v/v) Triton‐X‐100, 0.5% (w/v) sodium deoxycholate, 0.2% (w/v) SDS, 2 mmol/l Na₂EDTA, 10 mmol/l Hepes buffer (pH 7.5), 1xPhosSTOP (cocktail of phosphatase inhibitors, Roche) and 1xProtease inhibitors cocktail (Roche, Mannheim, Germany). Protein concentration was determined by the Bradford colorimetric assay (Pierce, Thermo Fisher Scientific Waltham, MA, USA). Immunoblot analysis of cell lysates was performed as previously described 30. To normalize protein load amounts, the blots were stripped (Re‐Blot Plus Mild Solution, Cat. No: 2502, Millipore, Thermo Fisher Scientific Waltham, MA, USA) and re‐probed with anti‐β‐tubulin monoclonal antibody (Cat. No: T4026; Sigma‐Aldrich).