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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.
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