Phosphatase inhibitor cocktail 3 phic

HEK293 cells were grown in DMEM medium supplemented 10% FBS (HyClone), 2 mM L-glutamin at 37 °C, 5% CO2. The cells were treated by CHX 20mkM or 6 and 12 uM (as indicated) of CKI-7 (Sigma). HEK293 cells were lysed in Lysis Buffer: 10 mM HEPES (pH 7.9) containing 5 mM MgCl, 0.5% Nonidet P-40, 0.45 M NaCl, 1 mM DTT, a protease inhibitor cocktail (PIC) (Roche) and a 1% Phosphatase inhibitor cocktail 3 (PhIC) (Sigma-Aldrich). The lysate was centrifuged at 10 000 rpm, 4 °C, for 10 min, and the supernatant was diluted 4-fold with the same buffer but without NaCl. The extract was treated with DNAse I (USB, 0.6 units/mL) and RNAse (Stratagene, 10 units/mL).
For cellular fractionation cells were lysed in FLB buffer (40 mM Tris-HCl, pH = 7,8; 100 mM NaCl; 2,5 mM MgCl2, 1 mM DTT; PIC; PhIC) on ice, grinded in Loose Dounce, centrifuged for 1 minute and supernatant was employed as the cytoplasmic fraction. The pellet was resuspended in Lysis Buffer, grinded in Thight Dounce, incubated 10 minutes on ice, centrifuged as mentioned above and diluted the same way.
The probes were equalized using Qubit Protein Assay Kit (ThermoFisher Scientific), mixed with 4X LB (200 mM Tris-HCl, pH = 6.8; 4% SDS; 40% Glycerol; ~0,01% Bromphenol blue; 100 mM DTT), and boiled for 10′.
Quantification of the Western blots was done using ImageJ gel analysis software (NIH, USA)⁴² .

HEK293 cells were grown in DMEM medium supplemented with 10% FBS (HyClone), 2 mM L-glutamine at 37°C, 5% CO₂. HEK293 cells were lysed in Lysis Buffer: 10 mM HEPES (pH 7.9) containing 5 mM MgCl, 0.5% Nonidet P-40, 0.45 M NaCl, 1 mM DTT, a protease inhibitor cocktail (PIC) (Roche) and a 1% Phosphatase inhibitor cocktail 3 (PhIC) (Sigma-Aldrich). The lysate was centrifuged at 10,000 rpm, 4°C, for 10 min, and the supernatant was diluted fourfold with the same buffer but without NaCl. The extract was treated with DNAse I (Thermo Fisher Scientific; 0.6 units/ml) and RNase (Thermo Fisher Scientific; 10 units/ml).