FIG. 5.

Effect of HD domain mutations on subunit dissociation under native conditions. Conditioned media from 293T cells expressing secreted unmutated and mutated LNR-HD polypeptides (sHDs) were immunoprecipitated (IP) with anti-FLAG (α-FLAG) beads (top two panels) and analyzed following SDS-PAGE by Western blot analysis with the indicated antibodies. To better gauge whether the coprecipitation of the sHD subunits was affected by the mutations, a range of coprecipitated unmutated sHD subunits were run as a control. In addition to the sHD subunits, variable amounts of unprocessed sHD were detected in most of the conditioned media, including the normal control. Mutations inducing subunit dissociation of the HA-tagged HD-C subunit are denoted by stars. Comparable results were observed in parallel experiments in which immunoprecipitates were prepared with anti-HA (α-HA) beads (bottom pair of panels), and coprecipitation of the FLAG-tagged HD-N subunit was assessed by Western blotting.