Fig. 3. DPY-21 associates physically with components of the dosage compensation complex. (A) Western blot of extracts from wild-type or dpy-21(e428) mutant gravid hermaphrodites serially diluted by 1.3-fold and probed with N-terminal antibodies to DPY-21 and antibodies to the loading control SMC-1, a protein involved in chromosome cohesion. Full-length DPY-21 (210 kDa) was present in extracts from wild-type but not dpy-21 mutant animals. A 60 kDa protein was variably detected in the dpy-21 mutant extract (asterisk). The apparent molecular weight of this protein is slightly larger than the 43 kDa protein predicted from the location of the e428 nonsense mutation at codon 394. The blot was also probed with antibodies to MIX-1, a protein involved in dosage compensation and chromosome condensation. Equivalent levels of MIX-1 in both mutant and wild-type extracts provide one example that dpy-21 mutations do not alter the stability of other dosage compensation proteins. (B) DPY-21 antibodies specifically precipitated SDC-3 from wild-type embryonic extracts (lane 3). SDC-3 was not immunoprecipitated by the preimmune sera (lane 1) or in a mock immunoprecipitation reaction that lacked antibodies (lane 2), showing specificity of the IP reactions. (C) DPY-27 antibodies strongly precipitated DPY-21 (lane 4). The precipitation of DPY-21 was specific, given that DPY-21 was not precipitated by the preimmune sera (lane 1) or antibodies to CBP-1, a DNA-associated CREB-binding protein (lane 2). DPY-21 antibodies failed to precipitate DPY-27 (lane 5), and DPY-26 antibodies only weakly precipitated DPY-21 (lane 3). These immunoprecipitation experiments indicate that DPY-21 interacts biochemically with other dosage compensation components but its association with the complex is probably not as robust as that of other members.