Figure 6.

Effect of 1,25(OH)₂D3 on VDR-RXR-α complex formation in cultured HuLM cells. A, HuLM cells were serum starved for 20 hours and treated with increasing doses of 1,25(OH)₂D3 (0, 10, 100, and 1000 nm) for 48 hours. Cell lysates prepared from these 1,25(OH)₂D3-treated cells were described in Materials and Methods. Cell lysates (1 mg) from each treatment condition were subjected to immunoprecipitation with polyclonal anti-RXR-α (top panel) or anti-VDR (bottom panel) antibodies, and immunoprecipitates were analyzed by Western blot using anti-VDR (top panel) or anti-RXR-α (bottom panel) antibodies. B, The above cell lysates were used to verify the expression of both VDR and RXR-α by Western blot analyses. C, Immunofluorescence analyses were performed using HuLM cells treated with increasing doses of 1,25(OH)₂D3 for 48 hours, as described above. Cells were fixed, permeabilized, and then incubated with anti-VDR, and anti-RXR-α antibodies, followed by incubation with CY3-conjugated (red) or FITC-conjugated (green) secondary antibodies, as appropriate. Nuclei of cells were monitored by DAPI (blue). Pictures were captured at ×200 magnification.