Prox1 is associated with LSD1/NuRD complex and directly interacts with LSD1.
(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 in vitro 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 E. coli BL21(DE3) and purified using Glutathione-Sepharose beads. Beads with bound GST-Prox1 proteins were then incubated in vitro translated LSD1 and LSD1 pulled down was detected using Western blot. GST was used as negative control.