FIGURE 3.

Co-localization analysis of Arr3 and L-PGDS by confocal immunofluorescence microscopy. A, HEK293 cells were transiently transfected with Arr3-GFP and L-PGDS-HA constructs and were treated with vehicle (upper panel) or 5 μm PGH₂ (lower panel) for the indicated times to stimulate L-PGDS enzymatic activity. Cells were then fixed and prepared as described under “Experimental Procedures.” Arr3-GFP is shown in green, whereas L-PGDS was visualized using anti-HA monoclonal and Alexa Fluor 546-conjugated anti-mouse IgG antibodies (red). Overlays of staining patterns (c, g, k, and o) and the corresponding pixel fluorograms (d, l, h, p) are also shown (x axis, intensity of L-PGDS pixels; y axis, intensity of Arr3-GFP pixels). A high degree of co-localization is revealed by a diagonal distribution (at 45°) of the dots on the fluorogram, whereas a lack of co-localization is characterized by two distinct populations with a minimal overlap of dots distributed toward the x and y axes, respectively. Scale bars, 10 μm. B, MG-63 cells were treated with vehicle (upper panel) or 100 μm SeCl₄ (lower panel) to inhibit PGDS enzymatic activity for 15 min. Cells were then fixed and prepared as described under “Experimental Procedures.” Arr3 was visualized using anti-Arr3 monoclonal antibody and Alexa Fluor 488-conjugated anti-mouse IgG antibodies (green, a and e), whereas L-PGDS was revealed with anti-L-PGDS polyclonal and Alexa Fluor 546-conjugated anti-rabbit IgG antibodies (red, b and f). Overlays of staining patterns (c and g) and the corresponding pixel fluorograms (d and h) are shown (x axis, intensity of L-PGDS pixels; y axis, intensity of Arr3 pixels). Scale bars, 10 μm.