Institution: NIH Library Sign In as Member / Individual

This Article Abstract Full Text Services Alert me to new issues of the journal Request Copyright Permission

Supporting Information

Files in this Data Supplement:

Exponentially growing 50 ml cultures of wild-type (NJK28), esa1-L245P (LPY3500) (20), vid21D (NJK1042), eaf7D (NJK1254), yng2D (NJK1482), eaf5D (NJK1259), htz1D (NJK1527) and swr1D (NJK1665) strains (see Table 4) were collected by centrifugation, resuspended into 37°C medium, and incubated at 37°C for 4 h. Cultures were pelleted, washed once in HSB b^o [45 mM Hepes-KOH pH 7.4/150 mM NaCl/10% glycerol/1 mM EDTA/0.5% Nonidet P-40/complete EDTA-free protease inhibitor mixture (Roche Diagnostics)] and resuspended in 300 m l HSB b^o. Cells were subjected to glass bead disruption at 4°C and centrifuged at 14,000 rpm in an Eppendorf 5417R for 10 min, and the supernatant was isolated. A total of 200 m g of each sample was resolved on a 15% SDS polyacrylamide gel. The gel was soaked in 62.5 mM Tris·Cl pH 6.8/2.3% SDS for 30 min and transferred overnight to nitrocellulose in Transfer b^o (25 mM ethanolamine/20% methanol, buffered to pH 7.5 with glycine). Immunoblotting with rabbit anti-hyperacetylated histone H4 (Penta) antibody (Upstate Biotechnology 06-946) followed by enhanced chemiluminescence was performed as described (1). Membranes were reprobed with anti-Cdc28 antibodies (kind gift from R. Deshaies, California Institute of Technology, Pasadena) to verify equal protein loading after 10 min at 75°C in stripping solution (75 mM Tris·Cl, pH 7.5/0.1% 2-mercaptoethanol).

1. Le Masson, I., Yu, D. Y., Jensen, K., Chevalier, A., Courbeyrette, R., Boulard, Y., Smith, M. M. & Mann, C. (2003) Mol. Cell. Biol. 23, 6086-6102.