Immunoblot analysis of tumor suppressor gene and oncogene products. (A) Comparison of U937 and RU cells for steady-state levels of the tumor suppressor gene products RB, p53, and p21. After cell lysis in RIPA buffer, 50 μg of total proteins were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes. Indicated proteins were detected by means of specific antibodies (1 μg/mL), and revealed by ECL after treatment with horseradish peroxidase-conjugated second antibody. The NB-E cell line was included as control for endogenous p53 protein production. (B) Effect of TPA on the production of c-Myc, c-Fos, and c-Jun oncoproteins in U937 and RU cells. Cultures (107 cells) were grown in the presence (+) or absence (−) of TPA (50 ng/mL) for 36 hours and processed for immunobloting as described in (A), using antibodies specific for the indicated proteins.
