Heme induces Bach2 degradation. (A) Heme-induced degradation of endogenous Bach2. The 18-81 pre-B cells were treated with 5μM heme or without heme for the indicated periods (minutes) in the presence of Chx. A Western blotting analysis of whole cell extracts was performed with antibodies against the indicated proteins. (B) Determination of the half-life of Bach2. The band intensities in panel A were measured by a densitometric analysis (National Institutes of Health imaging analysis). Open circle indicate treatment with Chx, and open triangles indicate treatment with Chx and 5μM heme. (C) Heme-induced degradation of endogenous Bach2 in mouse splenic B cells. Purified mouse splenic B220-positive B cells were stimulated with 20 μg/mL LPS in the presence or absence of 20μM heme for the indicated periods. A Western blotting analysis of whole-cell extracts was performed with antibodies against the indicated proteins (top). Coomassie blue staining of gels indicated that heme did not cause any gross change in the overall protein levels (bottom).