Fig 12.

Mus81 does not self-associate when chromatin bound. (A) Formaldehyde-fixed cells (WDHY2456) expressing Myc- and HA-tagged Mus81 were lysed, and whole-cell extract (WCE) was isolated. Centrifugation separated soluble proteins (SOL) from the chromatin-containing pellet (PEL). Protein from each fraction was prepared by trichloroacetic acid precipitation and separated by SDS-PAGE prior to transfer to nitrocellulose. The membrane was probed using anti-Mus81, anti-3-phosphoglycerate kinase, and anti-histone H3 antibodies. (B) Co-chromatin immunoprecipitation analysis. Cells were treated as described for panel A, and the pellets were washed three times in fresh lysis buffer, sonicated, and cleared of precipitated proteins and debris prior to immunoprecipitation using anti-HA, anti-Myc, and anti-Mus81 antibodies. Immunoprecipitated samples were separated on an SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed using anti-Mus81, anti-Myc, and anti-HA antibodies. Lane numbers represent unnumbered lanes from left to right, respectively, as follows: 1 and 3, WDHY3268; 2 and 4, WDHY3269 antibody specificity controls; 5 to 14 WDHY3267. For both panel A and panel B, membranes were stripped prior to each subsequent blot.