TABLE 2.
ChIP assays
| Parameter | Result for cell line with doxycycline/nocodazole treatmentb
|
||||||
|---|---|---|---|---|---|---|---|
| Foxi1-V5
|
Pac2-Tet
|
Foxi1-V5, +/+ | Pac2-Tet, +/+ | Blocked beads, −/− | |||
| +/− | +/− | +/− | +/− | ||||
| Antibody | Anti-V5 | Anti-GFP | Anti-V5 | Anti-GFP | Anti-V5 | Anti-V5 | |
| Amt of input DNA (μg) | 170.7 | 170.7 | 229.6 | 229.6 | 208 | 265.8 | 0 |
| Amt of immunoprecipitated DNA (μg) | 0.424 | 0.186 | 0.165 | 0.178 | 0.304 | 0.184 | 0.177 |
| Amt of enriched DNA (μg)a | 0.247 | 0.009 | 0 | 0.001 | 0.127 | 0.007 | 0 |
Immunoprecipitated DNA plus eluted DNA from preblocked protein A agarose. To reduce the background level of preblocked, sonicated herring sperm DNA, immunoprecipitated DNA was purified using the QIAquick PCR purification kit (Qiagen).
Quantified DNA recovery under various conditions. FoxI1-V5 fusion containing cells were tested under induced (with doxycycline) conditions compared to the cell line Pac2-Tet which is identical except for the absence of the FoxI1 fusion protein. The comparison was made between DNA precipitated by the appropriate monoclonal antibody (anti-V5) and an inappropriate control antibody (anti-GFP). The tests were also conducted in cells arrested in mitosis by nocodazole to demonstrate that FoxI1 can precipitate DNA when chromatin is condensed.