Western analysis and nuclear/cytoplasmic fractionation

For nuclear/cytoplasmic fractionation, 1 × 10⁷ C33a cells were treated with 30 μM compound for 6 hrs. The cells were washed with PBS and lysed in 1000 μL of cytoplasmic extraction buffer (10 mM KCl, 10 mM HEPES, pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol) and incubated on ice for 20 min. 50 μL of 10% NP-40 was added to each lysate, vortexed and then centrifuged at 12,000 × g for 10 min. The cytosolic fraction was collected and the pellet containing the nuclei suspended in 50 μL of nuclear extraction buffer (0.4 M NaCl, 20 mM HEPES, pH 7.9, 1 mM EDTA). The nuclear fraction was incubated for 30 min on ice. The samples were spun at 14,000 × g for 10 min and the supernatants were collected. To concentrate the cytoplasmic fraction, 500 μL methanol, and 200 μL chloroform were added and the samples were vortexed, centrifuged for 10 min at 14,000 × g, and the upper and lower layer were discarded. The solid interphase was collected and suspended in 50 μL of lysis buffer. Equal cell equivalents were loaded on a gel and electrophoresed. For whole cell lysates, C33a cells were lysed with a modified RIPA buffer and subjected to SDS-PAGE electrophoresis. The gels were transferred to nitrocellulose and blots probed with antibodies for p53 (Santa Cruz, antibody FL-303), lamin A/C, and calnexin (Santa Cruz Biotechnology).