Western blot analysis of samples from ChIP experiments, showing specificity of the IP obtained with the I12 anti-HOXB4 monoclonal antibody. (A) Formaldehyde cross-linked chromatin was prepared from clone 4 of KLS-EML-HOXB4 cells, as described in supplemental Methods (ChIP-chip), and incubated with the I12 antihuman HOXB4 rat monoclonal antibody that had been previously bound to magnetic beads (Dynabeads) conjugated to antirat IgG2a antibody. The IP lane represents the material that remained bound to the magnetic beads after repeated washings. Eluates 1 to 8 represent the supernatants from the serial washings of the magnetically bound beads. The HOXB4 protein was specifically immunoprecipitated using the I12 monoclonal anti-HOXB4 antibody, as shown by the presence of the HOXB4 protein and the absence of actin in the IP lane. Some residual HOXB4 protein was observed in the first eluate, indicating that binding was not 100% efficient. (B) Using an antirat IgG2a isotype control antibody, HOXB4 protein is not observed in the IP lane, further documenting the specificity of the I12 monoclonal anti-HOXB4 antibody used for ChIP. Similar results were obtained when chromatin extracts from clones 11 and 17 of KLS-EML-HOXB4 cells were used.