Fig. 3.
Fig. 3. PLZF associates with the VDR DBD in vitro by means of the BTB/POZ domain. / (A) Schematic diagram of the GST-VDR fusions used to assess an in vitro interaction between PLZF and VDR. These included the full-length VDR, the LBD (amino acids 110-427), the hinge region (amino acids 97-189), the DBD (amino acids 14-110), and GST alone. The VDR derivatives were affinity purified after expression in Escherichia coli on GST Sepharose. The expression levels of the GST-VDR derivatives were assessed by SDS-PAGE analysis of expressed proteins (data not shown). (B) PLZF interacts selectively in vitro with full-length GST-VDR and GST-VDRDBD (lanes 3 and 6, respectively) but not with GST-VDRLBD, GST-VDRhinge, or the orphan receptor HNF4DBD fused to GST (lanes 4, 5, and 7). PLZF was translated in the presence of 35S-methionine and incubated at 4°C with the various VDR derivatives depicted in panel A. Interacting complexes were washed and subjected to SDS-PAGE analysis, dried, and exposed to film. Similar amounts of specific protein were used in each experiment. Lane 1 represents 10% of the labeled PLZF input (I). PLZF runs slightly differently in the input lane because of the lack of BSA (which was added to stabilize interactions in the other lanes). Approximately 5 × 10−13 mM PLZF was used in each reaction. (C) Schematic diagram of PLZF derivatives. PLZFΔBTB/POZ (Δ amino acids 1-162), PLZFΔRD2 (Δ amino acids 199-313), and fBTB/POZ (flag-tagged amino acids 1-162) were used in subsequent in vitro GST pull-down assays. (D) The BTB/POZ domain is necessary and sufficient for PLZF interaction with VDR. GST-VDR on GST agarose beads (lane 3) was incubated with 35S-labeled, in vitro–translated PLZF, PLZFΔBTB/POZ, PLZFΔRD2, or fBTB/POZ at 4°C. Resulting complexes were washed and analyzed by SDS-PAGE and autoradiography. None of the derivatives interacted with GST alone (lane 2). Approximately 5 × 10−13 mM translated protein was used in each reaction. Pull-down experiments were done at least 3 times, and a representative film is shown.

PLZF associates with the VDR DBD in vitro by means of the BTB/POZ domain.

(A) Schematic diagram of the GST-VDR fusions used to assess an in vitro interaction between PLZF and VDR. These included the full-length VDR, the LBD (amino acids 110-427), the hinge region (amino acids 97-189), the DBD (amino acids 14-110), and GST alone. The VDR derivatives were affinity purified after expression in Escherichia coli on GST Sepharose. The expression levels of the GST-VDR derivatives were assessed by SDS-PAGE analysis of expressed proteins (data not shown). (B) PLZF interacts selectively in vitro with full-length GST-VDR and GST-VDRDBD (lanes 3 and 6, respectively) but not with GST-VDRLBD, GST-VDRhinge, or the orphan receptor HNF4DBD fused to GST (lanes 4, 5, and 7). PLZF was translated in the presence of 35S-methionine and incubated at 4°C with the various VDR derivatives depicted in panel A. Interacting complexes were washed and subjected to SDS-PAGE analysis, dried, and exposed to film. Similar amounts of specific protein were used in each experiment. Lane 1 represents 10% of the labeled PLZF input (I). PLZF runs slightly differently in the input lane because of the lack of BSA (which was added to stabilize interactions in the other lanes). Approximately 5 × 10−13 mM PLZF was used in each reaction. (C) Schematic diagram of PLZF derivatives. PLZFΔBTB/POZ (Δ amino acids 1-162), PLZFΔRD2 (Δ amino acids 199-313), and fBTB/POZ (flag-tagged amino acids 1-162) were used in subsequent in vitro GST pull-down assays. (D) The BTB/POZ domain is necessary and sufficient for PLZF interaction with VDR. GST-VDR on GST agarose beads (lane 3) was incubated with 35S-labeled, in vitro–translated PLZF, PLZFΔBTB/POZ, PLZFΔRD2, or fBTB/POZ at 4°C. Resulting complexes were washed and analyzed by SDS-PAGE and autoradiography. None of the derivatives interacted with GST alone (lane 2). Approximately 5 × 10−13 mM translated protein was used in each reaction. Pull-down experiments were done at least 3 times, and a representative film is shown.

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