Figure 3

Distribution of H2A.Z-2 and H2A.Z-1 within chromatin fractions. (A) S1, SE and P chromatin fractions were generated from HEK 293 cells stably expressing Flag epitope tagged H2A.Z-2 and H2A.Z-1, resolved by sodium dodecyl sulphate-PAGE and analysed by Western blot. The antibodies used for Western blotting are indicated on the right. The anti-H2A.Z antibody recognizes both isoforms in their endogenous (arrow 1) and Flag-tagged (arrow 2) forms. Probing with the anti-H3 Tri Me K4 antibody indicates that this modification is more enriched within the P fraction and therefore does not partition equally among the fractions. Total histone H4 was used as a loading control and CM indicates chicken erythrocyte histone marker. The trends seen were consistent across multiple experimental replicates. (B) 4% native acrylamide gel of purified DNA from the S1, SE and P chromatin fractions used in A. The S1 fraction contains mononucleosomes (M) with approximate DNA length 150 bp, while the SE and P fractions contain chromatin composed of mononucleosomes, dinucleosomes (Di), trinucleosomes (Tri), tetranucleosomes (Te) and longer chromatin. M is CFO-1 cut pBR322 DNA marker.