After transfection for 48 hours with p27kip1 siRNA or nonsilencing siRNA, cultured cells were trypsinized and pelleted. Proteins were extracted by incubating cells for 30 minutes at 4°C in buffer containing 1% Triton X-100, 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 rabbit polyclonal anti-p27kip1 diluted 1:200 in blocking buffer. Blots were rinsed three times for 10 minutes with 0.1% Triton X-100, then reblocked and exposed for 1 hour to horseradish peroxidase (HRP)–conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:10,000 in blocking solution. The same blots were probed with rabbit anti-nonmuscle myosin (Biomedical Technologies, Inc.) diluted 1:200 to control for protein load. After a thorough wash, peptides were detected with a chemiluminescent substrate (SuperSignal West Pico; Pierce, Rockford, IL). For quantification, films were digitally scanned (BDS-Image; Biological Detection System, Pittsburgh, PA). Scans were analyzed with NIH Image software version 1.61 (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD), and protein content was normalized according to nonmuscle myosin protein content. These experiments were repeated at least three times.