Nuclear and cytoplasmic protein extraction kit

Cells were lysed for total protein extraction using a radio immunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl buffer, 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium deoxycholate) in the presence of Protease Inhibitor Cocktail and PhoSTOP Phosphatase Inhibitor Cocktail (both from Roche, Basel, Switzerland) for 30 min, then cleared by centrifugation at 4°C. Nuclear extracts were isolated with a Nuclear and Cytoplasmic Protein Extraction Kit (Bioteke, Beijing, China), according to the manufacturer's instructions. Both total and nuclear extracts were quantified using a BCA assay kit (KeyGEN, Nanjing, China), then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. These membranes were the blotted with appropriate primary antibodies and horseradish peroxidase-coupled secondary antibodies, and visualized using chemiluminescence (DNR Bio-Imaging Systems, Jerusalem, Israel) and x-ray films. For Western Blot analysis, β-tubulin and lamin B were used as loading control. Antibodies were obtained from various sources, including Sigma–Aldrich (anti-β-tubulin, anti-β-catenin and anti-CCNG2), Abcam (Cambridge, MA, USA) (anti-CCNG2 and anti-Runx2), Sangon Biotech (anti-ALP and anti-lamin B) and Santa Cruz (anti-CCND1 and anti-c-Myc).

Nuclear and cytoplasmic extracts were isolated with a Nuclear and Cytoplasmic Protein Extraction Kit (Bioteke, Beijing, China) upon the manufacturer’s instructions. Both the extracts were quantified using a BCA assay kit (Pierce®, Thermo Scientific, USA). Simple western analysis was performed using the WES™ device (ProteinSimple, San Jose, CA, USA) upon the manufacturer’s protocols. In brief, 3 μL of proteins was loaded on the plate, labeled with a biotin reagent, and detected by chemiluminescence using streptavidin-horseradish peroxidase. The proportion of the protein of interest was then measured for its nuclear or cytoplasmic fraction, in comparison to the biotinylated ladder. Data were analyzed using Compass™ software (Version 4.0.0, ProteinSimple). Each protein peak was measured automatically and the median area under the peak was normalized to the LMNB1 for nuclear protein or the ACTB for cytoplasmic protein.