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Cellular localization of PanX1 in stable cell line overexpressing PanX-EGFP, (LNCaP-PanX1). A representative pair of adjacent cells (transmitted phase-contrast image is shown on the top left in A) was chosen for confocal imaging. Spatial pattern of PanX1 distribution visualized with EGFP florescence (excited by the 488-nm line of argon laser; the fluorescence was captured at wavelengths 505–530 nm) was generally similar to that in transiently transfected LNCaP cells (see Fig. 2) and consisted of plasma membrane and intracellular pools with distinct expression of PanX1 in junctional regions (A, top left). A fluorescence confocal image of one cell from this pair taken at higher magnification and thinner confocal optical slice (<0.5 μm) revealed that intracellular distribution of PanX1 is consistent with its localization in the ER; interconnected tubules forming a dense network are clearly seen in the confocal image of EGFP fluorescence (A, middle image). This was further confirmed by staining the cell with ER-specific marker BODIPY 558/568 brefeldin A (excited by the 543-nm line of HeNe laser; the fluorescence was captured at wavelengths >560 nm). The overlay of the EGFP and BODIPY brefeldin A fluorescence confocal images (A, bottom right) allows clear distinction between plasmalemmal (green) and ER (yellow) localization of PanX1. (A, bottom left) An enlarged boxed region (from A, bottom left) after rotation by 90°. Note that confocal images in top, middle, and bottom images were taken at different focusing (Z positions). Similar protocol was applied to another LNCaP cell. Confocal images of the EGFP and BODIPY brefeldin A fluorescence, as well as their overlay, are presented as indicated (B, top). The enlarged boxed regions taken at higher magnification are presented below each image after rotation by 90°, respectively (B, bottom). Note that the confocal images shown were obtained as a result of averaging of four sequential images taken in multitrack configuration of the confocal scanner followed by low-pass filtering (7 × 7 pixels; LSM 510 software) to improve the signal-to-noise ratio.

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