Pierce bicinchoninic acid bca assay

Cells were transfected for 24 hours, and if required, treated with drugs (thalidomide, 100 μM/lenalidomide, 10 μM) for 24 hours. In experiments with drug treatments, a time 0 (0.1% DMSO) control was included. Cells were washed in ice-cold PBS, then lysed with RIPA lysis buffer (Sigma) and proteasomal inhibitors. Samples were left on ice for 15 minutes, briefly vortexed, and returned to the ice for 15 minutes. Lysed cells were centrifuged for 15 minutes at 16,000 × g at 4°C. The supernatant containing protein was carefully removed and quantification of total protein concentration was assessed using the Pierce bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA). Samples were run on 4%−12% Nupage Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA) and nitrocellulose membranes were probed with antibodies against GAPDH (1:2000) (Sigma, St. Louis, MO), CRBN (1:500) (HPA045910, Sigma Prestige, St. Louis, MO), AGO2 (1:500) (Sigma Prestige, St. Louis, MO), or SALL4 (1:50) (abcam, Cambridge, MA). Blotting was detected by incubating with IR-secondary antibodies and scanning the membrane at the appropriate wavelengths (Licor, Lincoln, NE).

On the day the gel was to subject to electrophoresis, the protein content of the samples was determined using a Pierce bicinchoninic acid (BCA) assay (Thermo Fisher Waltham, MA, USA). Samples were diluted five-fold and loaded in duplicate into a 96 well plate. For comparison, 8 standards of known protein concentration were loaded in triplicate. A spectrophotometer (Molecular Devices, Sunnyvale, CA, USA), and associated Softmax Pro software (Molecular Devices, Sunnyvale, CA, USA) were used to determine the volume of supernatant fluid needed to load a consistent mass of 20 μg total protein per well.