HCECs were lysed and subjected, with modification, to subcellular fractionation followed by Western blot analysis with six PKC isozyme-specific antibodies. Serum-starved HCECs were treated with 20 ng/mL EGF or 1 μM PDBu for 30 seconds to 30 minutes; untreated HCECs were the control. Subsequent steps were performed as described earlier.
17 20 Cells were washed twice with ice-cold PBS and scraped into a homogenization buffer. Homogenization buffer components were 25 mM Tris HCl (pH 7.4), 2 mM EDTA, 10 mM β-mercaptoethanol, 10% glycerol, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were allowed to swell for 10 minutes and then were homogenized with approximately 30 strokes in a tight-fitting pestle (Dounce homogenizer; Bellco Glass Co., Vineland, NJ). The homogenates were centrifuged at 500
g for 5 minutes, and the low-speed supernatant was centrifuged at 100,000
g for 30 minutes. High-speed supernatant constituted the cytosolic fraction. The high-speed pellet was extracted in ice-cold homogenization buffer containing 1% Triton X-100 for 30 to 60 minutes. The Triton-soluble component (membrane fraction) was separated from the Triton-insoluble material (cytoskeletal fraction) by centrifugation at 100,000
g for 15 minutes. The cytoskeletal fraction was resuspended in the same buffer and dispersed by sonication. The low-speed pellet containing nuclei and unbroken cells was resuspended in a nuclear buffer. Nuclear buffer components were 25 mM Tris HCl (pH 7.4), 3 mM MgCl
2, 1 mM PMSF, 10 mM β-mercaptoethanol, and 0.05% Triton X-100. To remove contaminating membrane components, the low-speed pellet homogenate was centrifuged for 5 minutes at 500
g, resuspended in the nuclear buffer without Triton X-100, layered over 45% sucrose, and centrifuged at 1900
g for 30 minutes. Purified nuclei were resuspended in the homogenization buffer containing 1% Triton X-100 for 30 to 60 minutes. The small amount of insoluble material was removed by centrifugation at 100,000
g for 15 minutes at 4°C, and the supernatant was the nuclear fraction. Protein concentration was measured with a bicinchoninic acid assay (Micro BCA protein assay kit; Pierce Biotechnology, Rockford, IL).