Prox1 is associated with LSD1/NuRD complex and directly interacts with LSD1.
2013-04-23T02:29:17Z (GMT) by
<p>(A) Identification of Prox1-associated proteins using immunoprecipitation and mass spectrometry (IP-MS). HEK293T cells were transfected with plasmid expressing FLAG-tagged Prox1 and Prox1-associated proteins were immunoprecipitated using anti-FLAG monoclonal antibodies. Cells transfected with empty vector were processed in parallel as negative control. Precipitated proteins were resolved on denaturing SDS-PAGE and silver-stained. Bands exclusively found in FLAG-Prox1 samples were excised and identified using MS. Positions of bands corresponding to Prox1 and multiple LSD1/NuRD complex components are indicated. (B) Association of exogenous Prox1 with LSD1/NuRD complex in HEK293T cells. HEK293T cells transfected with plasmid expressing FLAG-tagged Prox1 or empty vector were subjected to co-immunoprecipitation assay using anti-FLAG monoclonal antibodies. Co-immunoprecipitated proteins were detected in Western blot using antibodies to LSD1/NuRD complex components as indicated. One tenth of cell lysate before co-immunoprecipitation was used as input control. (C) Association of endogenous Prox1 with LSD1/NuRD complex in HepG2 cells. HepG2 cells were subjected to co-immunoprecipitation assay using anti-Prox1 antibodies. Co-immunoprecipitated HNF4α and LSD1/NuRD complex components were detected in Western blot using corresponding antibodies as indicated. (D) Prox1 directly interacts with <i>in vitro</i> translated LSD1 in GST pulldown assay. Schematic representation of Prox1 domain organization is depicted (top). GST-fused repression (aa 1–337), central (aa 335–570) and Prospero/homeo (aa 544–738) domains of Prox1 were expressed in <i>E. coli</i> BL21(DE3) and purified using Glutathione-Sepharose beads. Beads with bound GST-Prox1 proteins were then incubated <i>in vitro</i> translated LSD1 and LSD1 pulled down was detected using Western blot. GST was used as negative control.</p>