FIGURE 7.

Effects of RXRα phosphorylation on nucleocytoplasmic kinetics of RXRα in HPK1A and HPK1ARas cells. FLIP methodology was used to assess nucleocytoplasmic kinetics. HPK1A cells were transfected with RXRαWT-GFP (A–E). Following transfection, live cells were treated with either vehicle (Veh) or 1,25(OH)₂D₃ (1,25D3) and nucleocytoplasmic trafficking was measured using confocal microscopy (see “Experimental Procedures”). Nuclear area was selected and photobleached (A), and other regions of interests measured but not photobleached (B, 1–3). C, time course showing (i) an unbleached nucleus of a neighboring cell and (ii) a cell with a bleached nucleus. The normalized fluorescent intensity of the unbleached and bleached nuclei above is shown in D. The dissociation curve of vehicle and 1,25(OH)₂D₃-treated cells are shown in E. The bound fractions of HPK1A cells transfected with RXRαWT-GFP (F) or HPK1ARas cells transfected with either RXRαWT-GFP (G) or RXRαmut-GFP (H) are similarly shown. Values are mean ± S.D. of at least 10 cells per treatment. Asterisks indicate a significant difference in bound fraction between vehicle (Veh) and 1,25(OH)₂D₃ (1,25D3) cells. A p value < 0.05 was considered significant.