Cultured cells were trypsinized and pelleted, and proteins were extracted by incubating cells for 30 minutes at 4°C in buffer containing 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO), 250 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma-Aldrich), followed by homogenization and centrifugation. Protein content was quantified by spectrophotometry. Equal protein was loaded on 4% to 12% Bis-Tris gels for SDS-PAGE. Peptides were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), and nonspecific binding was blocked by incubation overnight at 4°C in 5% nonfat milk diluted in PBS. Membranes were incubated for 2 hours with primary antibody diluted in blocking buffer. Antibody dilutions, prepared in blocking buffer, were as follows: p27kip1, 1:200; p21cip1, 1:100; p15INK4b, 1:100; cyclin-D1, 1:200; cyclin-E , 1:100; cyclin-A, 1:100; cyclin-B1, 1:200; CDK2, 1:100; CDK4, 1:50; and nonmuscle myosin, 1:200. Blots were rinsed for 10 minutes three times with 0.1% Triton X-100, then reblocked and exposed for 1 hour to HRP-conjugated donkey anti-rabbit, -mouse, or -goat IgG, diluted 1:10,000 in blocking solution. The same blots were probed with rabbit anti-nonmuscle myosin to control for protein load. After a thorough wash, peptides were detected using chemiluminescent substrate (SuperSignal West Pico; Pierce, Rockford, IL). For quantification, films were digitally scanned (BDS-Image; Biological Detection Systems, Pittsburgh, PA), scans were analyzed by computer (NIH Image ver. 1.61; W. Rasband, National Institutes of Health, Bethesda, MD; available by FTP from zippy.nimh.nih.gov or on floppy disc from NTIS, Springfield, VA), and protein content was normalized according to nonmuscle myosin protein content.