VDR and PLZF interact in U937 nuclear extracts.

(A) Immunoblot for expression of PLZF in nuclear extracts. Nuclear extracts from the tetracycline-regulated stable line U937-PLZF45 were prepared from cells grown for 36 hours in the presence (− PLZF) or absence (+ PLZF) of tetracycline and immunoblotted for PLZF expression by using an anti-PLZF monoclonal antibody. (B) Coimmunoprecipitation of PLZF and VDR from nuclear extracts. Immunoprecipitations were done by using nuclear extracts from U937-PLZF45 cells with either anti-VDR or anti-PLZF monoclonal antibodies and protein A/G agarose. Precipitated complexes were washed in propidium iodide buffer containing 200 mM KCl, separated by SDS-PAGE, and immunoblotted for either PLZF (lanes 1-3) or VDR (lanes 4-6). Preimmune serum and protein A/G beads were used as a negative control immunoprecipitation (lanes 3 and 6). (C) Coimmunoprecipitation of PLZF and mSin3A. Antibodies directed against PLZF (lanes 4 and 5) but not those against VDR (lanes 1 and 2) coimmunoprecipitated Sin3A from PLZF45 extracts. Coimmunoprecipitation was performed as in panel B, except that the complexes were immunoblotted for mSin3A with an antibody that cross-reacts with human Sin3A. Preimmune serum and protein A/G beads were used as a control (lanes 3 and 6). (D) Coimmunoprecipitation of PLZF and VDR in SKNO cells. Lysates of these cells were subjected to direct immunoblotting (lane 2) or blotting after precipitation with PLZF antibody (lane 4) or an isotype control (lane 3). A positive control (lane 1), confirming the efficacy of the antibody and size of the precipitated species, consisted of 25 ng purified baculovirus-derived VDR (VDR^baculo). Direct immunoblotting was done with 100 μg lysate from SKNO-1 cells. PI indicates preimmune serum; IP, immunoprecipitation; Lys, lysate.