FIGURE 16.
Effects of heme on the subcellular localization and steady-state levels of Rev-erbβ. A, HEK293 cells transiently transfected with pcDNA3.1(+)-FLRev-erbβ, pcDNA3.1(+)-FLRev-erbβ-H568F, or pcDNA3.1(+)-FLRev-erbβ-H568F/C384A were fractionated into cytosolic (Cyto) and nuclear (Nuc) compartments, and the fractions were immunoblotted with Rev-erbβ LBD antibody; 25 ng of recombinant FLRev-erbβHGS, used as a positive control (lane R). The cytosolic and nuclear fractions are indicated by black bars above the corresponding lanes, and duplicate biological replicates (Rep.) are shown for each fraction. B, the purity of cytosolic and nuclear fractions assessed by probing the membranes described in A with antibodies against lamin B1 (nuclear envelope), NCoR1 (nucleus), and Hsp90 (cytosol). A representative blot is shown for each protein with duplicate biological replicates. C, the effect of altering intracellular heme on the distribution and steady-state level of endogenous Rev-erbβ. HEK293 cells were incubated with 1 mm SA for 42 h or left untreated as a control (indicated by black bars above the appropriate lanes). The blot is arranged as described in A, with duplicate biological replicates shown for each fraction; 5 ng of FLRev-erbβMGC as a positive control (lane R). Blots assessing the purity of the fractions are also depicted.