Journal: Molecular and Cellular Biology
Article Title: CENP-A, -B, and -C Chromatin Complex That Contains the I-Type ?-Satellite Array Constitutes the Prekinetochore in HeLa Cells
Figure Lengend Snippet: Bulk chromatin and centromeric chromatin were solubilized by MNase digestion of HeLa nuclei in 0.3 M NaCl. (A) Centromeric proteins CENP-A, -B, and -C were solubilized by MNase digestion. Isolated HeLa nuclei (2 × 10 8 ) were suspended with 1 ml of WB containing 0.3 M NaCl (sample a in lane 1 and sample c in lanes 4 to 6) or 0.6 M NaCl (sample b in lanes 2 and 3). Sample c was digested with 60 U of MNase per ml for 10 min at 37°C. Soluble and insoluble materials from each sample were separated by centrifugation. ACA beads were added to the supernatant of sample c and incubated overnight at 4°C. Pellets were resuspended in 1 ml of SDS buffer by extensive sonication and 5 μl of each sample was separated by SDS-7.5% (for CENP-B and CENP-C) or 12.5% (for CENP-A) PAGE, and centromeric proteins were detected by immunostaining with ACA serum. Lane 1, supernatant fraction of a; lane 2, supernatant fraction of b; lane 3, pellet fraction of b; lane 4, supernatant fraction of c before addition of ACA beads; lane 5, supernatant fraction of c after addition of ACA beads; lane 6, pellet fraction of c. Lane M, marker centromeric proteins, CENP-A, CENP-B, and CENP-C. (B) Size distribution of DNA fragments from bulk chromatin after MNase digestion. HeLa nuclei were digested with MNase to various extents. The fragmented DNA in the soluble fractions was extracted with phenol and electrophoresed through 1% agarose gel. DNA was detected with ethidium bromide staining. Lane 1, 20 U/ml for 2 min (40 U/ml × min, sample 1); lane 2, 20 U/ml for 4 min (80 U/ml × min, sample 2); lane 3, 40 U/ml for 5 min (200 U/ml × min, sample 3); lane 4, 80 U/ml for 45 min (3,600 U/ml × min, sample 4). Positions of the DNA size markers are indicated at the left. (C) Detection of core histones and CENP-A in each fraction. Soluble (sup.) and insoluble (pellet) fractions were subjected to SDS-12.5% PAGE, and the separated core histones were stained with Coomassie brilliant blue (upper panel). The proteins were transferred to a membrane and immunolabeled with ACA serum (AK) (lower panel). Lane M in the lower panel is a recombinant CENP-A marker protein. Lanes 1 to 4 correspond to samples 1 to 4 of the soluble (sup.) fractions, and lanes 5 to 8 to samples 1 to 4 of the pellet fractions.
Article Snippet: The nuclear suspension was digested with MNase (Roche Diagnostics) at 37°C after addition of CaCl2 to a final concentration of 2 mM.
Techniques: Isolation, Western Blot, Centrifugation, Incubation, Sonication, Polyacrylamide Gel Electrophoresis, Immunostaining, Marker, Agarose Gel Electrophoresis, Staining, Immunolabeling, Recombinant