Complete phosphatase inhibitors

HEK293T cells (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM/F12; Thermo Fischer Scientific) supplemented with 10% foetal calf serum (FCS;Thermo Fischer Scientific) at 37°C, 5% CO₂. For immunoblot experiments 100,000 cells were plated in a 24-well plate (Nunc) overnight and constructs transfected with Lipofectamine 2000 (Thermo Fischer Scientific). For immunofluorescence (IF) experiments, cells were plated overnight and transfected with Lipofectamine 2000. After 24 hours, cells were replated onto a 384 well CellCarrier plate (PerkinElmer). The cells were either lysed or fixed 48 hours post-transfection. Lysis was performed using TNE-TX buffer (10mM Tris, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, cOmplete protease inhibitors (Sigma), cOmplete phosphatase inhibitors (Sigma)), incubating for 20 min on ice and centrifugation for 5 min at 4°C. Alternatively, lysis for signalling experiments was performed using TNE-SDS buffer (10mM Tris, 150 mM NaCl, 1mM EDTA, 1% SDS, cOmplete protease inhibitors (Sigma), cOmplete phosphatase inhibitors (Sigma)), incubating 10 min on ice, before sonicating and centrifugation for 10 min. For IF experiments, cells were washed in ice cold PBS and fixed in 4% PFA (Sigma) for 10 min.