FIGURE 6.

Effects of RXRα phosphorylation on VDR and RXR interaction in HPK1A and HPK1ARas cells. A, FRET was measured by acceptor photobleaching as described under “Experimental Procedures.” Cells were co-transfected with either VDR-CFP/RXRαWT-YFP or VDR-CFP/RXRαmut-YFP. Following transfection, cells were treated with either vehicle (Veh), 1,25(OH)₂D₃ (1,25D3), UO126 alone, or a combination of UO126 and 1,25(OH)₂D₃ (C–E). B, HPK1A cells were co-transfected with either CFP/YFP (negative control) or VDR-CFP/RXRαWT-YFP plasmids. Similarly, both HPK1A (F) and HPK1ARas (G) cells were grown and treated with either vehicle, 1,25(OH)₂D₃, UO126 alone, or a combination of UO126 and 1,25(OH)₂D₃. The cells were then fixed and co-stained with VDR-Cy3 and RXRα-Alexa-488 antibodies to detect both endogenous VDR and RXRα. FRET was measured by acceptor photobleaching as described under “Experimental Procedures.” Scatter plots show FRET measurement of HPK1A cells co-transfected with VDR-CFP/RXRαWT-YFP (C) or co-stained with VDR-cy3 and RXRα-Alexa-488 antibodies (F). Similarly, in HPK1ARas cells scatter plots show cells co-transfected with either VDR-CFP/RXRαWT-YFP (D), VDR-CFP/RXRαmut-YFP (E), or co-stained with VDR-cy3 and RXRα-Alexa-488 antibodies (G). Values are mean percentage dequenching ± S.D. of at least 10 cells per treatment condition. FRET baseline was set at 100%. Asterisks indicate a significant difference in interaction between 1,25(OH)₂D₃ treatment alone compared with vehicle-treated control. Open circles indicate a significant difference in interaction between 1,25(OH)₂D₃-treated cells alone compared with combined UO126 and 1,25(OH)₂D₃ treatment. A p value of p < 0.05 was considered significant.