Sustained Release of Prostaglandin E₂ in Fibroblasts Expressing Ectopically Cyclooxygenase 2 Impairs P2Y-Dependent Ca²⁺-Mobilization

Figure 3

Subcellular distribution of P2Y2, P2Y4, and P2Y6 receptors in MEFs. WT or KI MEFs were cultured and, after changing the medium, were maintained in the absence or presence of 1 μM DFU for 2 h. Cells were fixed with paraformaldehyde (4%; pH 7.2) and permeabilized with cold methanol at RT. After incubation with anti-P2Y2, anti-P2Y4 and anti-P2Y6 antibodies (1 : 500) overnight at 4°C, cells were visualized by confocal microscopy using a FITC-conjugated secondary Ab (Alexa-Fluor 488, 1 : 1000). Nuclei were stained with Hoechst 33258. Coverslips were mounted in Prolong Gold antifade reagent (Molecular Probes) and the intensity of the fluorescence was measured using Image J software (NIH, Bethesda, MD, USA). Results show the mean + SD of three experiments. versus the same condition in the WT cells.