Figure 3.

The R80W mutation does not affect binding to 1,25(OH)₂D₃ and RXR but significantly affects the subcellular localization of VDR^R80W (A) Relative VDR protein expression as determined by the density of the VDR bands from Western blotting analysis of VDR^WT and VDR^R80W CD4⁺ T cells activated in the absence (-) or presence (+) of 1,25(OH)₂D₃ (10 nM). The upper panel gives the density of VDR^WT (black circles) and VDR^R80W (white squares) normalized to the average density of the VDR^WT bands from control cells activated in the absence of 1,25(OH)₂D₃. The lower panel shows one representative Western blotting analysis out of five independent experiments of VDR and GAPDH (loading control) from control and patient cells. Mean ± SD (VDR^WT n = 13; VDR^R80W n = 1 repeated 5 times; *p < 0.05). (B–D) Subcellular localization of VDR^WT and VDR^R80W in CD4⁺ T cells activated in (B) the absence (-) or presence (+) of 1,25(OH)₂D₃ (10 nM) and in (C, D) the absence (-) or presence (+) of RA (1 µM) and 1,25(OH)₂D₃ (10 nM). The upper panels show the fraction of VDR located to the nucleus and the cytosol in grey and black columns, respectively, as determined by the density of the VDR bands from the Western blots. The lower panels show one representative Western blotting analysis of VDR and GAPDH (loading control) from the cytosolic (C) and nuclear (N) fraction of CD4⁺ T cells activated in the absence (-) or presence (+) of 1,25(OH)₂D₃ and RA as indicated. (B) Mean ± SD (VDR^WT n = 14; VDR^R80W n = 1 repeated 5 times; *p < 0.05). (C, D) Mean ± SD (VDR^WT n = 8; VDR^R80W n = 1 repeated 3 times; *p < 0.05). (E) Relative mRNA expression of CYP24A1 in VDR^WT (black circles) and VDR^R80W (white squares) CD4⁺ T cells activated in the absence (-) or presence (+) of RA (1 µM) and 1,25(OH)₂D₃ (10 nM). The expression levels are given as fold change normalized to the average expression level of CYP24A1 in control cells activated in the absence of RA and 1,25(OH)₂D₃. Mean ± SD (VDR^WT n = 6; VDR^R80W n = 1 repeated twice; *p < 0.05).