Figure 7

A) Structure of the formaldehyde sensitive amino acids and sensitive groups in biotin (arrows). B) Western blot analysis of two binding experiments where non-crosslinked nuclear extracts (lanes 1, 2, 3) and formaldehyde crosslinked chromatin (lanes 4, 5, 6) were tested. Input and supernatant fractions (lanes 1 and 2, 4 and 5 respectively) represent 1% of total material, while bound lanes (3 and 6) represent 25% of total material. The biotin-V5-tagged GATA-1 was detected with N6 anti-GATA-1 antibody (top panel). After stripping, the same membrane was incubated with streptavidin-HRP (lower panel). C) Western blot showing comparison of binding of two different extracts: crosslinked chromatin (lanes 1–3) and non-crosslinked nuclear extract (lanes 4–6) precipitated by anti-V5 agarose. The biotin-V5-tagged GATA-1 was detected with N6 anti-GATA-1 antibody (top panel), stripped and re-probed with streptavidin-HRP (lower panel). Input and supernatant fractions (lanes 1 and 3, 4 and 6 respectively) represent 1% of total material, while bound lanes (2 and 4) represent 50% of total material. Note that V5-tagged GATA-1 is completely depleted in both experiments as the supernatant lanes are empty.