5loading buffer

Cell lysate in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 10 μg/ml aprotinin) was mixed with 5 loading buffer (Fermentas, Waltham, MA, USA) and heated at 95 °C for 5 min. Samples were separated electrophoretically on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked and probed with: mouse anti-DAB2 (1:1,000; BD Biosciences, San Jose, CA, USA), or rabbit anti-β-actin (1:3,000; Cell Signaling Technology, Danvers, MA, USA) antibodies. Bound antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Santa Cruz, Dallas, TX, USA) at a dilution of 1:5,000 for 45 min at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences), and the membrane was exposed to X-ray film (Agfa, Mortsel, Belgium). Anti-β-actin antibody was used to confirm that loading was comparable between gel lanes. The density of each protein band was read and quantified using ImageJ software.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL pepstatin A, and 10 µg/mL aprotinin). Cell lysate equivalent to 50 μg protein was mixed with 5× loading buffer (Fermentas, Waltham, MA, USA) and heated at 95 °C for 5 min. Samples were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Membranes were blocked and probed with the following antibodies: mouse anti-E2F3 (1:500; Santa Cruz Biotechnology, SC-56665) and rabbit anti-β-actin (1:2000; Cell Signaling Technology, #4970S). Bound antibodies were detected with horseradish-peroxidase-conjugated anti-mouse (GeneTex, GTX213111-01, Irvine, CA, USA) and anti-rabbit (Cell Signaling Technology, #7074S) secondary antibodies at a dilution of 1:3000 for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Bioscience, Piscataway, NJ, USA), and the membrane was exposed to X-ray film (Agfa, Mortsel, Belgium). Anti-β-actin antibody was used to confirm comparable loading between samples. The density of each protein band was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).