![Fig. 5. PLZF and VDR comigrate in a DNA-bound complex. / (A) Endogenous VDR from U937-PLZF45 nuclear extracts binds to a VDRE with an altered mobility in the presence of PLZF. Nuclear extracts were prepared from U937-PLZF45 cells and incubated in the presence or absence of tetracycline (− PLZF or + PLZF). Then, 5μg nuclear extract was combined with a phosphorus 32–labeled VDRE probe, incubated for 20 minutes at room temperature, and electrophoresed on a nondenaturing polyacrylamide gel. The VDRE probe used in this assay was a DR-3 from the mouse OPN gene promoter. Lane 1 is free probe and lanes 2 to 7 show the specificity of the shifts observed. Lane 2 depicts the 4 major species found in U937 nuclear extracts without PLZF expression, which competed with unlabeled (10-fold excess) specific oligonucleotide (spec) (lane 3) but not a nonspecific oligonucleotide (non-spec) (a glucocorticoid response element [GRE]; lane 4). Lanes 5 through 7 show controls similar to those in lanes 2 to 4 but with use of extracts expressing PLZF. There was a significant change in mobility in the presence of PLZF (lane 5). This shift competed with the specific oligonucleotide (lane 6) and was not affected by the nonspecific oligonucleotide (lane 7). Lanes 8 through 11 show the effects of the addition of antibodies to VDR on the mobility shift. The presence of control IgG antibody had no effect on the shifted species (lane 8). The addition of anti-VDR antibody caused a loss of the major species (open arrow) and a supershift (asterisk) indicating that the complex contained VDR. In the presence of PLZF, another major species (solid arrow) was supershifted (2 asterisks) by anti-VDR antibodies (lane 10 versus lane 11). (B) PLZF is present in a DNA-bound complex containing VDR. EMSA was done as shown in panel A. The addition of anti-PLZF antibody did not affect the shift pattern observed when PLZF was absent (lanes 12 and 13 versus 19). The addition of anti-VDR antibodies confirmed the presence of VDR in a DNA-bound complex. VDR complexes were abrogated by the addition of anti-PLZF antibody (lanes 16 and 17; arrow), indicating that both PLZF and VDR exist in the bound species. The addition of preimmune serum did not affect the shifts in the absence (lane 18) or presence (lane 19) of PLZF.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/98/12/10.1182_blood.v98.12.3290/6/h82311804005.jpeg?Expires=1726830987&Signature=LcyNllEEfHd2Xxqw8ThJfSrF9sC6U8mzKXZdkkqZzlcO6JAKPLRl6V8ZIo5gwsZLS3QwzA8lPqt1EdbFhJrekluqJ12JivW6KN-kYjxMblscXoUwAmSD2p-nEL1yPsqrCc3PAUGv9pL5pDs5tTbgM34TZO~09WE4NvZZt1Vl2Y1nu2fcagvOLdLvGiG1QZeLp3JwfelFhQI-pRXtnsaVZZ3loyeub4keUZ9VZNgMs15NTpCzvFZPhoWaWLqRhTj2F01rNFfGHvXo7obAur9NcQtkwJWYo8kTHYuVcGKcdYYWgFxedN0cF9BBMsNbg9EnXEizuP0h57ZGwwZar-5k-g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PLZF and VDR comigrate in a DNA-bound complex.
(A) Endogenous VDR from U937-PLZF45 nuclear extracts binds to a VDRE with an altered mobility in the presence of PLZF. Nuclear extracts were prepared from U937-PLZF45 cells and incubated in the presence or absence of tetracycline (− PLZF or + PLZF). Then, 5μg nuclear extract was combined with a phosphorus 32–labeled VDRE probe, incubated for 20 minutes at room temperature, and electrophoresed on a nondenaturing polyacrylamide gel. The VDRE probe used in this assay was a DR-3 from the mouse OPN gene promoter. Lane 1 is free probe and lanes 2 to 7 show the specificity of the shifts observed. Lane 2 depicts the 4 major species found in U937 nuclear extracts without PLZF expression, which competed with unlabeled (10-fold excess) specific oligonucleotide (spec) (lane 3) but not a nonspecific oligonucleotide (non-spec) (a glucocorticoid response element [GRE]; lane 4). Lanes 5 through 7 show controls similar to those in lanes 2 to 4 but with use of extracts expressing PLZF. There was a significant change in mobility in the presence of PLZF (lane 5). This shift competed with the specific oligonucleotide (lane 6) and was not affected by the nonspecific oligonucleotide (lane 7). Lanes 8 through 11 show the effects of the addition of antibodies to VDR on the mobility shift. The presence of control IgG antibody had no effect on the shifted species (lane 8). The addition of anti-VDR antibody caused a loss of the major species (open arrow) and a supershift (asterisk) indicating that the complex contained VDR. In the presence of PLZF, another major species (solid arrow) was supershifted (2 asterisks) by anti-VDR antibodies (lane 10 versus lane 11). (B) PLZF is present in a DNA-bound complex containing VDR. EMSA was done as shown in panel A. The addition of anti-PLZF antibody did not affect the shift pattern observed when PLZF was absent (lanes 12 and 13 versus 19). The addition of anti-VDR antibodies confirmed the presence of VDR in a DNA-bound complex. VDR complexes were abrogated by the addition of anti-PLZF antibody (lanes 16 and 17; arrow), indicating that both PLZF and VDR exist in the bound species. The addition of preimmune serum did not affect the shifts in the absence (lane 18) or presence (lane 19) of PLZF.
