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Figure 7


Fig. 7. Coexpression of GNL3L increases the electrophoretic mobility of the DNA-bound ERR{gamma} protein complex and reduces its binding with SRC1 and SRC2. (A) The GNL3L effect on the binding of ERR{gamma} to DNA was examined by EMSA using ERE-containing probes and whole-cell lysates expressing the indicated recombinant proteins. Compared with the probe alone (lane 1) and the vector-transfected control sample (lane 2), ERR{gamma}-specific DNA-protein complex was identified in lane 3 (arrow b), competed by excess non-labeled probes (lane 4), and supershifted by anti-Myc antibody (lane 5, arrow a). Coexpression of GNL3L produces fast-moving complexes (lane 6, arrows d and e) that can be supershifted by anti-Myc antibody (lane 7, arrow c) but not by anti-HA antibody (lane 8). GNL3L itself cannot bind the ERE probe (lane 9). The intensity of the fast-moving complex d is reduced by a deletion of the ERR{gamma}-binding I-domain of GNL3L (lanes 10-12). (B) The fast-moving complex d and the slow-moving complex b were retrieved from the EMSA gel, fractionated in SDS-denaturing PAGE and analyzed for their ERR{gamma} ({alpha}-Myc), GNL3L ({alpha}-HA), SRC1, and SRC2 protein components by western blotting. Our results indicate that the increase in the electrophoretic mobility of the ERR{gamma}-DNA complex by GNL3L coexpression is due to a loss of SRC1 binding (arrow) and diminished SRC2 binding, rather than by protein cleavage of ERR{gamma}.





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