Fig. 7. Quantitative analysis of the surface distribution of tagged GFP TA constructs with the use of extracellular reagents. (A) MDCK cells expressing GFP-22-Nglyc were exposed to anti-opsin mAbs from both the apical and the basolateral compartments of a Transwell filter chamber, followed by Cy5-conjugated anti-mouse antibodies. After fixation and permeabilisation, they cells were labelled with rat anti-E cadherin followed by Cy3-conjugated anti-rat antibodies. Single confocal sections of the same field in an apical (a-d) and lateral (e-h) plane are shown (positions on the z axis correspond to the third and tenth section of panel B), visualised for total GFP (a,e), extracellular opsin tag (b,f) or cadherin (c,g). Acquisition with each filter and image processing was carried out under the same conditions in the apical and lateral plane. Note the absence of intracellular staining with the anti-opsin mAb in panel (f). (d) and (h) show the merged image from panels (a-c) and (e-g) respectively (GFP in green, opsin tag in red and cadherin in blue). The yellow colour in (d) indicates colocalisation between apical GFP and the extracellular opsin tag. The white segments in (h) indicate colocalisation of basolateral GFP, extracellular tag and cadherin. Bar, 10 µm. (B) The distributions of extracellular opsin tag and of cadherin were determined in Z series of confocal sections acquired on GFP-22-Nglyc- (left panel) and GFP-22-MutNglyc- (right panel) expressing cells, treated as in (A). , extracellular opsin tag, , E-cadherin. The average of eight determinations, with s.e.m. (bars), are shown. See Materials and Methods for details. (C) Western blot analysis of surface biotinylated MDCK cells. MDCK cells, expressing GFP-22-Nglyc (lanes 3, 4) or GFP-22-MutNglyc (lanes 5, 6), or non-transfected cells (lanes 1, 2) were biotinylated either from the apical (lanes 1, 3, 5) or the basolateral (lanes 2, 4, 6) compartment of Transwell filter chambers. Biotinylated proteins were collected with streptavidin beads and analysed by western blotting with anti-GFP (upper panel) or anti-cadherin (lower panel) antibodies. The bracket, arrow and arrowhead in the upper panel indicate the mature glycosylated, glycosylated Endo-H-sensitive and unglycosylated forms of GFP-22-Nglyc, respectively. The mature glycosylated form is distributed both on the apical and basolateral surface, whereas the non glycosylated form of GFP-22-Nglyc, as well as GFP-22-MutNgly and cadherin, are detected exclusively on the basolateral side. Numbers on the left indicate the position and size (kDa) of markers (Bio-Rad).

Return to article