Figure 7. RIPK1 increases VDR cytoplasmic localization.
293T cells were transfected with Flag-VDR and Flag-RIPK1 constructs. Thirty-six hours post-transfections, cells were treated with EtOH or 10−7 M 1,25D3 for 24 hours. Whole cell, cytosolic, and nuclear extracts were prepared and subjected to Western blot analyses with indicated antibodies. Anti-HSP60 and PARP-1 Western blots were included as loading controls for cytoplasmic and nuclear proteins, respectively. The intensity of Western blot bands was quantified by ImageJ (NIH). VDR signals were first normalized with cognate loading controls. Cytoplasmic and nuclear VDR were further normalized with corresponding whole-cell VDR and plotted as relative VDR signals. Bar graphs represent four independent experiments (n=4) and error bars have been calculated as mean ± standard error of the mean (SEM). *p<0.05, **<0.005.