|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 1. Translocation of PKC
-EGFP upon stimulation of P2X7 receptors. RAW 264.7 cells were seeded onto glass-bottomed dishes, cultured for 2 days in the presence of RANKL and transfected with plasmids encoding PKC-EGFP. At 2 days following transfection, cells were bathed in HEPES-buffered, bicarbonate-free medium and observed using confocal microscopy. (A) In the absence of BzATP, PKC-EGFP was uniformly distributed throughout the cytosol. Notably, there was no fluorescence in the nuclei. (B) Treatment of the same osteoclast-like cell with the P2X7-receptor agonist BzATP (150 µM) caused redistribution of PKC-EGFP to the plasma membrane (representative of 68 out of 77 cells examined). The image was obtained 
30 seconds following the addition of BzATP. The lower panels in A and B are reconstructed z-stack images of the osteoclast-like cell shown above. PKC translocated exclusively to the upper (basolateral) membrane. Blue horizontal lines indicate the location of the confocal images shown above. (C,D) PKC translocation was quantified by using line profiles of fluorescence intensity, indicated by white diagonal lines in A and B. Fluorescence within 5 µm of the edge of the cell was considered to be membrane associated and the intervening region was considered cytosolic. Lines were positioned to avoid nuclei. Localization was quantified from the average pixel intensity in the membrane-associated regions (Fm, outlined by pink rectangles) and the average pixel intensity in the cytosol (Fc, outlined by blue rectangle), with values of the ratio Fm:Fc exceeding 1 taken as indicating membrane localization. For the line scans shown, the membrane-localization ratio was 0.43 in the absence and 4.8 in the presence of BzATP. Data shown are representative of responses of cells from 14 independent transfections (as summarized in Table 1).
|
